Pneumococcal capsular saccharide conjugate vaccine

ABSTRACT

The present invention is in the field of pneumococcal capsular saccharide conjugate vaccines. Specifically, an immunogenic composition for infants is provided comprising a multivalent Streptococcus pneumoniae vaccine comprising 2 or more capsular saccharide conjugates from different serotypes, wherein the composition comprises a serotype 22F saccharide conjugate. Such a vaccine may be used in infant populations to reduce the incidence of elderly pneumococcal disease such as exacerbations of COPD and/or IPD.

PRIORITY

The present application is a continuation application from U.S.application Ser. No. 14/459,312 filed Aug. 14, 2014, which is acontinuation application from U.S. application Ser. No. 12/097,611 filedJun. 16, 2008 pursuant to 35 U.S.C. § 371 as a United States NationalPhase Application of International Patent Application Serial No.PCT/EP2006/069979 filed Dec. 20, 2006, which claims priority from GreatBritain Application No. 0526232.4 filed in the United Kingdom on Dec.22, 2005; Great Britain Application No. 0607087.4 filed in the UnitedKingdom on Apr. 7, 2006; Great Britain Application No. 0607088.2 filedin the United Kingdom on Apr. 7, 2006; Great Britain Application No.0609902.2 filed in the United Kingdom on May 18, 2006; Great BritainApplication No. 0620336.8 filed in the United Kingdom on Oct. 12, 2006;Great Britain Application No. 0620337.6 filed in the United Kingdom onOct. 12, 2006; Great Britain Application No. 0620815.1 filed in theUnited Kingdom on Oct. 19, 2006; Great Britain Application No. 0620816.9filed in the United Kingdom on Oct. 19, 2006; and from PCT ApplicationNo. GB2006/004634 filed in the United Kingdom on Dec. 12, 2006, thecontents of each of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to an improved Streptococcus pneumoniaevaccine.

SEQUENCE LISTING

This application contains sequences, listed in an electronic SequenceListing entitled VB62170C1_US_Seq_Listing_S25, 2 KB in size, createdusing Patent-In 3.5 on Mar. 3, 2015, the contents and sequences of whichare hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

Children less than 2 years of age do not mount an immune response tomost polysaccharide vaccines, so it has been necessary to render thepolysaccharides immunogenic by chemical conjugation to a proteincarrier. Coupling the polysaccharide, a T-independent antigen, to aprotein, a T-dependent antigen, confers upon the polysaccharide theproperties of T dependency including isotype switching, affinitymaturation, and memory induction.

However, there can be issues with repeat administration ofpolysaccharide-protein conjugates, or the combination ofpolysaccharide-protein conjugates to form multivalent vaccines. Forexample, it has been reported that a Haemophilus influenzae type bpolysaccharide (PRP) vaccine using tetanus toxoid (TT) as the proteincarrier was tested in a dosage-range with simultaneous immunization with(free) TT and a pneumococcal polysaccharide-TT conjugate vaccinefollowing a standard infant schedule. As the dosage of the pneumococcalvaccine was increased, the immune response to the PRP polysaccharideportion of the Hib conjugate vaccine was decreased, indicating immuneinterference of the polysaccharide, possibly via the use of the samecarrier protein (Dagan et al., Infect Immun. (1998); 66: 2093-2098)

The effect of the carrier-protein dosage on the humoral response to theprotein itself has also proven to be multifaceted. In human infants itwas reported that increasing the dosage of a tetravalent tetanus toxoidconjugate resulted in a decreased response to the tetanus carrier (Daganet al. supra). Classical analysis of these effects of combinationvaccines have been described as carrier induced epitopic suppression,which is not fully understood, but believed to result from an excessamount of carrier protein (Fattom, Vaccine 17: 126 (1999)). This appearsto result in competition for Th-cells, by the B-cells to the carrierprotein, and B-cells to the polysaccharide. If the B-cells to thecarrier protein predominate, there are not enough Th-cells available toprovide the necessary help for the B-cells specific to thepolysaccharide. However, the observed immunological effects have beeninconsistent, with the total amount of carrier protein in some instancesincreasing the immune response, and in other cases diminishing theimmune response.

Hence there remain technical difficulties in combining multiplepolysaccharide conjugates into a single, efficacious, vaccineformulation.

Streptococcus pneumoniae is a Gram-positive bacterium responsible forconsiderable morbidity and mortality (particularly in the young andaged), causing invasive diseases such as pneumonia, bacteraemia andmeningitis, and diseases associated with colonisation, such as acuteOtitis media. The rate of pneumococcal pneumonia in the US for personsover 60 years of age is estimated to be 3 to 8 per 100,000. In 20% ofcases this leads to bacteraemia, and other manifestations such asmeningitis, with a mortality rate close to 30% even with antibiotictreatment.

Pneumococcus is encapsulated with a chemically linked polysaccharidewhich confers serotype specificity. There are 90 known serotypes ofpneumococci, and the capsule is the principle virulence determinant forpneumococci, as the capsule not only protects the inner surface of thebacteria from complement, but is itself poorly immunogenic.Polysaccharides are T-independent antigens, and can not be processed orpresented on MHC molecules to interact with T-cells. They can however,stimulate the immune system through an alternate mechanism whichinvolves cross-linking of surface receptors on B cells.

It was shown in several experiments that protection against invasivepneumococci disease is correlated most strongly with antibody specificfor the capsule, and the protection is serotype specific.

Streptococcus pneumoniae is the most common cause of invasive bacterialdisease and Otitis media in infants and young children. Likewise, theelderly mount poor responses to pneumococcal vaccines [Roghmann et al.,(1987), J. Gerontol. 42:265-270], hence the increased incidence ofbacterial pneumonia in this population [Verghese and Berk, (1983)Medicine (Baltimore) 62:271-285].

The major clinical syndromes caused by S. pneumoniae are widelyrecognized and discussed in all standard medical textbooks (Fedson D S,Muscher D M. In: Plotkin S A, Orenstein W A, editors. Vaccines. 4rthedition. PhiladelphiaWB Saunders Co, 2004a: 529-588). For instance,Invasive pneumococcal disease (IPD) is defined as any infection in whichS. pneumoniae is isolated from the blood or another normally sterilesite (Musher D M. Streptococcus pneumoniae. In Mandell G L, Bennett J E,Dolin R (eds). Principles and Practice of Infectious diseases (5th ed).New York, Churchill Livingstone, 2001, p 2128-2147). Chronic obstructivepulmonary disease (COPD) is recognised as encompassing severalconditions (airflow obstruction, chronic bronchitis, bronchiolitis orsmall airways disease and emphysema) that often coexist. Patients sufferexacerbations of their condition that are usually associated withincreased breathlessness, and often have increased cough that may beproductive of mucus or purulent sputum (Wilson, Eur Respir J 200117:995-1007). COPD is defined physiologically by the presence ofirreversible or partially reversible airway obstruction in patients withchronic bronchitis and/or emphysema (Standards for the diagnosis andcare of patients with chronic obstructive pulmonary disease. AmericanThoracic Society. Am J Respir Crit Care Med. 1995 November; 152 (5 Pt2):S77-121). Exacerbations of COPD are often caused by bacterial (e.g.pneumococcal) infection (Sethi S, Murphy T F. Bacterial infection inchronic obstructive pulmonary disease in 2000: a state-of-the-artreview. Clin Microbiol Rev. 2001 April; 14(2):336-63).

It is thus an object of the present invention to develop an improvedformulation of a multiple serotype Streptococcus pneumoniaepolysaccharide conjugate vaccine.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 Bar chart showing 11 valent conjugate immunogenicity in elderlyRhesus monkeys. The lighter bars represent the GMC after twoinoculations with 11 valent conjugate in aluminium phosphate adjuvant.The darker bars represent the GMC after two inoculations with 11 valentconjugate in adjuvant C.

FIG. 2 Bar chart showing memory B cells for PS3 after inoculation withthe 11 valent conjugate in adjuvant C or aluminium phosphate adjuvant.

FIG. 3 Bar chart showing anti polysaccharide 19F immunogenicity inBalb/C mice for the 4-valent plain polysaccharides and the 4-valent dPlyconjugates.

FIG. 4 Bar chart showing anti polysaccharide 22F immunogenicity inBalb/C mice for the 4-valent plain polysaccharides and the 4-valent PhtDconjugates.

FIG. 5 Bar chart showing anti-22F IgG response in Balb/c mice

FIG. 6 Bar chart showing anti-22F opsono-phagocytosis titres in Balb/cmice.

FIG. 7 Bar chart comparing IgG responses induced in young C57B1 miceafter immunisation with 13 Valent conjugate vaccine formulated indifferent adjuvants.

FIG. 8 Bar chart showing the protective efficacy of different vaccinecombinations in a monkey pneumonia model.

FIG. 9 Bar chart showing anti PhtD IgG response in Balb/c mice afterimmunisation with 22F-PhtD or 22F-AH-PhtD conjugates.

FIG. 10 Protection against type 4 pneumococcal challenge in mice afterimmunisation with 22F-PhtD or 22F-AH-PhtD.

DESCRIPTION OF THE INVENTION

The present invention provides an immunogenic composition for infantscomprising a multivalent Streptococcus pneumoniae vaccine comprising 2or more (e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15) capsular saccharideconjugates from different serotypes, wherein the composition comprises a22F saccharide conjugate.

Although childhood infection from pneumococcus serotype 22F is not verycommon, the inventors believe that the presence of 22F in a childhoodpneumococcal vaccine will be advantageous in inducing herd immunity inthe population such that the onset of serious elderly disease caused bythis serotype (such as pneumonia and/or invasive pneumococcal disease(IPD) and/or exacerbations of chronic obstructive pulmonary disease(COPD)) may be prevented or reduced in severity. For the purposes ofthis invention, “immunizing a human host against exacerbations of COPD”or “treatment or prevention of exacerbations of COPD” or “reduction inseverity of COPD exacerbations” refers to a reduction in incidence orrate of COPD exacerbations (for instance a reduction in rate of 0.1,0.5, 1, 2, 5, 10, 20% or more) or a reduction in severity of COPDexacerbations as defined above, for instance within a patient groupimmunized with the compositions or vaccines of the invention.

Thus in one embodiment a method of preventing an elderly human host fromhaving a pneumococcal disease caused by Streptococcus pneumoniaeserotype 22F infection (or reducing its severity) is provided comprisingadministering to an infant human host (or an infant human population) animmunoprotective dose of the immunogenic composition or the vaccine ofthe invention. A use of the immunogenic composition or vaccine of theinvention in the manufacture of a medicament for the prevention orreduction in severity of a disease caused by serotype 22F Streptococcuspneumoniae infection in elderly human patients, wherein animmunoprotective dose of the composition or vaccine is administered toan infant human (or infant population).

In one embodiment the immunogenic composition comprises Streptococcuspneumoniae capsular saccharide conjugates from serogroups 19A and 19F,optionally wherein 19A is conjugated to a first bacterial toxoid and 19Fis conjugated to a second bacterial toxoid.

The term capsular saccharide includes capsular polysaccharides andoligosaccharides derivable from the capsular polysaccharide. Anoligosaccharide contains at least 4 sugar residues.

The term bacterial toxoid includes bacterial toxins which areinactivated either by genetic mutation, by chemical treatment or byconjugation. Suitable bacterial toxoids include tetanus toxoid,diphtheria toxoid, pertussis toxoid, bacterial cytolysins orpneumolysin. Mutations of pneumolysin (Ply) have been described whichlower the toxicity of pneumolysin (WO 90/06951, WO 99/03884). Similarly,genetic mutations of diphtheria toxin which lower its toxicity are known(see below). Genetically detoxified analogues of diphtheria toxininclude CRM197 and other mutants described in U.S. Pat. Nos. 4,709,017,5,843,711, 5,601,827, and 5,917,017. CRM197 is a non-toxic form of thediphtheria toxin but is immunologically indistinguishable from thediphtheria toxin. CRM197 is produced by C. diphtheriae infected by thenontoxigenic phase p197tox-created by nitrosoguanidine mutagenesis ofthe toxigenic carynephage b (Uchida et al Nature New Biology (1971) 233;8-11). The CRM197 protein has the same molecular weight as thediphtheria toxin but differs from it by a single base change in thestructural gene. This leads to a glycine to glutamine change of aminoacid at position 52 which makes fragment A unable to bind NAD andtherefore non-toxic (Pappenheimer 1977, Ann Rev, Biochem. 46; 69-94,Rappuoli Applied and Environmental Microbiology September 1983p₅₆₀-564).

The first and second bacterial toxoids may be the same or different.Where the first and second bacterial toxoids are different, it is meantthat they have a different amino acid sequence.

For example, 19A and 19F may be conjugated to tetanus toxoid and tetanustoxoid; diphtheria toxoid and diphtheria toxoid; Crm197 and CRM197,pneumolysin and pneumolysin, tetanus toxoid and diphtheria toxoid;tetanus toxoid and CRM197; tetanus toxoid and pneumolysin; diphtheriatoxoid and tetanus toxoid; diphtheria toxoid and CRM197, diphtheriatoxoid and pneumolysin; CRM197 and tetanus toxoid, CRM197 and diphtheriatoxoid; CRM197 and pneumolysin; Pneumolysin and tetanus toxoid;pneumolysin and diphtheria toxoid; or pneumolysin and CRM197respectively.

In an embodiment, in addition to S. pneumoniae saccharide conjugate of22F (and optionally 19A and 19F), the immunogenic composition furthercomprises conjugates of S. pneumoniae capsular saccharides 4, 6B, 9V,14, 18C and 23F.

In an embodiment, in addition to S. pneumoniae saccharide conjugate of22F (and optionally 19A and 19F), the immunogenic composition furthercomprises conjugates of S. pneumoniae capsular saccharides 1, 4, 5, 6B,7F, 9V, 14, 18C and 23F.

In an embodiment, in addition to S. pneumoniae saccharide conjugate of22F (and optionally 19A and 19F), the immunogenic composition furthercomprises conjugates of S. pneumoniae capsular saccharides 1, 4, 5, 6B,7F, 9V, 14, 18C, 22F and 23F.

In an embodiment, in addition to S. pneumoniae saccharide conjugate of22F (and optionally 19A and 19F), the immunogenic composition furthercomprises conjugates of S. pneumoniae capsular saccharides 1, 3, 4, 5,6B, 7F, 9V, 14, 18C, 22F and 23F.

In an embodiment, in addition to S. pneumoniae saccharide conjugate of22F (and optionally 19A and 19F), the immunogenic composition furthercomprises conjugates of S. pneumoniae capsular saccharides 1, 3, 4, 5,6A, 6B, 7F, 9V, 14, 18C, 22F and 23F.

Typically the Streptococcus pneumoniae vaccine of the present inventionwill comprise capsular saccharide antigens (preferably conjugated),wherein the saccharides are derived from at least ten serotypes of S.pneumoniae. The number of S. pneumoniae capsular saccharides can rangefrom 10 different serotypes (or “V”, valences) to 23 different serotypes(23V). In one embodiment there are 10, 11, 12, 13, 14 or 15 differentserotypes. In another embodiment of the invention, the vaccine maycomprise conjugated S. pneumoniae saccharides and unconjugated S.pneumoniae saccharides. Preferably, the total number of saccharideserotypes is less than or equal to 23. For example, the invention maycomprise 10 conjugated serotypes and 13 unconjugated saccharides. In asimilar manner, the vaccine may comprise 11, 12, 13, 14 or 16 conjugatedsaccharides and 12, 11, 10, 9 or 7 respectively, unconjugatedsaccharides.

In one embodiment the multivalent pneumococcal vaccine of the inventionwill be selected from the following serotypes 1, 2, 3, 4, 5, 6A, 6B, 7F,8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and33F, although it is appreciated that one or two other serotypes could besubstituted depending on the age of the recipient receiving the vaccineand the geographical location where the vaccine will be administered.For example, an 10-valent vaccine may comprise polysaccharides fromserotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F. An 11-valentvaccine may also include saccharides from serotype 3. A 12 or 13-valentpaediatric (infant) vaccine may also include the 10 or 11 valentformulation supplemented with serotypes 6A and 19A, or 6A and 22F, or19A and 22F, or 6A and 15B, or 19A and 15B, or 22F and 15B, whereas a13-valent elderly vaccine may include the 11 valent formulationsupplemented with serotypes 19A and 22F, 8 and 12F, or 8 and 15B, or 8and 19A, or 8 and 22F, or 12F and 15B, or 12F and 19A, or 12F and 22F,or 15B and 19A, or 15B and 22F. A 14 valent paediatric vaccine mayinclude the 10 valent formulation described above supplemented withserotypes 3, 6A, 19A and 22F; serotypes 6A, 8, 19A and 22F; serotypes6A, 12F, 19A and 22F; serotypes 6A, 15B, 19A and 22F; serotypes 3, 8,19A and 22F; serotypes 3, 12F, 19A and 22F; serotypes 3, 15B, 19A and22F; serotypes 3, 6A, 8 and 22F; serotypes 3, 6A, 12F and 22F; orserotypes 3, 6A, 15B and 22F.

The composition in one embodiment includes capsular saccharides derivedfrom serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (preferablyconjugated). In a further embodiment of the invention at least 11saccharide antigens (preferably conjugated) are included, for examplecapsular saccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14,18C, 19F and 23F. In a further embodiment of the invention, at least 12or 13 saccharide antigens are included, for example a vaccine maycomprise capsular saccharides derived from serotypes 1, 3, 4, 5, 6A, 6B,7F, 9V, 14, 18C, 19A, 19F and 23F or capsular saccharides derived fromserotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F,although further saccharide antigens, for example 23 valent (such asserotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F,18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by theinvention.

The vaccine of the present invention may comprise protein D (PD) fromHaemophilus influenzae (see e.g. EP 0594610). Haemophilus influenzae isa key causative organism of otitis media, and the present inventors haveshown that including this protein in a Streptococcus pneumoniae vaccinewill provide a level of protection against Haemophilus influenzaerelated otitis media (reference POET publication). In one embodiment,the vaccine composition comprises protein D. In one aspect, PD ispresent as a carrier protein for one or more of the saccharides. Inanother aspect, protein D could be present in the vaccine composition asa free protein. In a further aspect, protein D is present both as acarrier protein and as free protein. Protein D may be used as a fulllength protein or as a fragment (WO0056360). In a further aspect,protein D is present as a carrier protein for the majority of thesaccharides, for example 6, 7, 8, 9 or more of the saccharides may beconjugated to protein D. In this aspect, protein D may also be presentas free protein.

The vaccine of the present invention comprises one, two or moredifferent types of carrier protein. Each type of carrier protein may actas carrier for more than one saccharide, which saccharides may be thesame or different. For example, serotypes 3 and 4 may be conjugated tothe same carrier protein, either to the same molecule of carrier proteinor to different molecules of the same carrier protein. In oneembodiment, two or more different saccharides may be conjugated to thesame carrier protein, either to the same molecule of carrier protein orto different molecules of the same carrier protein.

Any Streptococcus pneumoniae capsular saccharides present in theimmunogenic composition of the invention may be conjugated to a carrierprotein independently selected from the group consisting of TT, DT,CRM197, fragment C of TT, PhtD, PhtDE fusions (particularly thosedescribed in WO 01/98334 and WO 03/54007), detoxified pneumolysin andprotein D. A more complete list of protein carriers that may be used inthe conjugates of the invention is presented below.

The carrier protein conjugated to one or more of the S. pneumoniaecapsular saccharides in the conjugates present in the immunogeniccompositions of the invention is optionally a member of thepolyhistidine triad family (Pht) proteins, fragments or fusion proteinsthereof. The PhtA, PhtB, PhtD or PhtE proteins may have an amino acidsequence sharing 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with asequence disclosed in WO 00/37105 or WO 00/39299 (e.g. with amino acidsequence 1-838 or 21-838 of SEQ ID NO: 4 of WO 00/37105 for PhtD). Forexample, fusion proteins are composed of full length or fragments of 2,3 or 4 of PhtA, PhtB, PhtD, PhtE. Examples of fusion proteins arePhtA/B, PhtA/D, PhtA/E, PhtB/A, PhtB/D, PhtB/E. PhtD/A. PhtD/B, PhtD/E,PhtE/A, PhtE/B and PhtE/D, wherein the proteins are linked with thefirst mentioned at the N-terminus (see for example WO01/98334).

Where fragments of Pht proteins are used (separately or as part of afusion protein), each fragment optionally contains one or more histidinetriad motif(s) and/or coiled coil regions of such polypeptides. Ahistidine triad motif is the portion of polypeptide that has thesequence HxxHxH where H is histidine and x is an amino acid other thanhistidine. A coiled coil region is a region predicted by “Coils”algorithm Lupus, A et al (1991) Science 252; 1162-1164. In an embodimentthe or each fragment includes one or more histidine triad motif as wellas at least one coiled coil region. In an embodiment, the or eachfragment contains exactly or at least 2, 3, 4 or 5 histidine triadmotifs (optionally, with native Pht sequence between the 2 or moretriads, or intra-triad sequence that is more than 50, 60, 70, 80, 90 or100% identical to a native pneumococcal intra-triad Pht sequence—e.g.the intra-triad sequence shown in SEQ ID NO: 4 of WO 00/37105 for PhtD).In an embodiment, the or each fragment contains exactly or at least 2, 3or 4 coiled coil regions. In an embodiment a Pht protein disclosedherein includes the full length protein with the signal sequenceattached, the mature full length protein with the signal peptide (forexample 20 amino acids at N-terminus) removed, naturally occurringvariants of Pht protein and immunogenic fragments of Pht protein (e.g.fragments as described above or polypeptides comprising at least 15 or20 contiguous amino acids from an amino acid sequence in WO00/37105 orWO00/39299 wherein said polypeptide is capable of eliciting an immuneresponse specific for said amino acid sequence in WO00/37105 orWO00/39299).

In particular, the term “PhtD” as used herein includes the full lengthprotein with the signal sequence attached, the mature full lengthprotein with the signal peptide (for example 20 amino acids atN-terminus) removed, naturally occurring variants of PhtD andimmunogenic fragments of PhtD (e.g. fragments as described above orpolypeptides comprising at least 15 or 20 contiguous amino acids from aPhtD amino acid sequence in WO00/37105 or WO00/39299 wherein saidpolypeptide is capable of eliciting an immune response specific for saidPhtD amino acid sequence in WO00/37105 or WO00/39299 (e.g. SEQ ID NO: 4of WO 00/37105 for PhtD).

If the protein carrier is the same for 2 or more saccharides in thecomposition, the saccharides could be conjugated to the same molecule ofthe protein carrier (carrier molecules having 2 more differentsaccharides conjugated to it) [see for instance WO 04/083251].Alternatively the saccharides may each be separately conjugated todifferent molecules of the protein carrier (each molecule of proteincarrier only having one type of saccharide conjugated to it).

Examples of carrier proteins which may be used in the present inventionare DT (Diphtheria toxoid), TT (tetanus toxoid) or fragment C of TT, DTCRM197 (a DT mutant) other DT point mutants, such as CRM176, CRM228, CRM45 (Uchida et al J. Biol. Chem. 218; 3838-3844, 1973); CRM 9, CRM 45,CRM102, CRM 103 and CRM107 and other mutations described by Nicholls andYoule in Genetically Engineered Toxins, Ed: Frankel, Maecel Dekker Inc,1992; deletion or mutation of Glu-148 to Asp, Gln or Ser and/or Ala 158to Gly and other mutations disclosed in U.S. Pat. No. 4,709,017 or4,950,740; mutation of at least one or more residues Lys 516, Lys 526,Phe 530 and/or Lys 534 and other mutations disclosed in U.S. Pat. No.5,917,017 or 6,455,673; or fragment disclosed in U.S. Pat. No.5,843,711, pneumococcal pneumolysin (Kuo et al (1995) Infect Immun 63;2706-13) including ply detoxified in some fashion for example dPLY-GMBS(WO 04081515, PCT/EP2005/010258) or dPLY-formol, PhtX, including PhtA,PhtB, PhtD, PhtE and fusions of Pht proteins for example PhtDE fusions,PhtBE fusions (WO 01/98334 and WO 03/54007), (Pht A-E are described inmore detail below) OMPC (meningococcal outer membrane protein—usuallyextracted from N. meningitidis serogroup B—EP0372501), PorB (from N.meningitidis), PD (Haemophilus influenzae protein D—see, e.g., EP 0 594610 B), or immunologically functional equivalents thereof, syntheticpeptides (EP0378881, EP0427347), heat shock proteins (WO 93/17712, WO94/03208), pertussis proteins (WO 98/58668, EP0471177), cytokines,lymphokines, growth factors or hormones (WO 91/01146), artificialproteins comprising multiple human CD4+ T cell epitopes from variouspathogen derived antigens (Falugi et al (2001) Eur J Immunol 31;3816-3824) such as N19 protein (Baraldoi et al (2004) Infect Immun 72;4884-7) pneumococcal surface protein PspA (WO 02/091998), iron uptakeproteins (WO 01/72337), toxin A or B of C. difficile (WO 00/61761).

Nurkka et al Pediatric Infectious Disease Journal. 23(11):1008-14, 2004Nov. described an 11 valent pneumococcal vaccine with all serotypesconjugated to PD. However, the present inventors have shown thatopsonophagocytic activity was improved for antibodies induced withconjugates having 19F conjugated to DT compared with 19F conjugated toPD. In addition, the present inventors have shown that a greater crossreactivity to 19A is seen with 19F conjugated to DT. It is therefore afeature of the composition of the present invention that serotype 19F isconjugated to a bacterial toxoid, for example TT, pneumolysin, DT or CRM197. In one aspect, serotype 19F is conjugated to DT. It is also afeature of the invention that serotype 19A is conjugated to a bacterialtoxoid, for example TT, pneumolysin, DT or CRM 197. The remainingsaccharide serotypes of the immunogenic composition may all beconjugated to one or more carrier proteins that are not DT (i.e. only19F is conjugated to DT), or may be split between one or more carrierproteins that are not DT and DT itself. In one embodiment, 19F isconjugated to DT or CRM 197 and all of the remaining serotypes areconjugated to PD. In a further embodiment, 19F is conjugated to DT orCRM 197, and the remaining serotypes are split between PD, and TT or DTor CRM 197. In a further embodiment, 19F is conjugated to DT or CRM 197and no more than one saccharide is conjugated to TT. In one aspect ofthis embodiment, said one saccharide is 18C or 12F. In a furtherembodiment, 19F is conjugated to DT or CRM 197 and no more than twosaccharides are conjugated to TT. In a further embodiment, 19F isconjugated to DT or CRM 197, and the remaining serotypes are splitbetween PD, TT and DT or CRM 197. In a further embodiment, 19F isconjugated to DT or CRM 197, and the remaining serotypes are splitbetween PD, TT and pneumolysin. In a further embodiment, 19F isconjugated to DT or CRM 197, and the remaining serotypes are splitbetween PD, TT and CRM 197. In a further embodiment, 19F is conjugatedto DT or CRM197 and the remaining serotypes are split between PD, TT,pneumolysin and optionally PhtD or PhtD/E fusion protein. In a furtherembodiment, 19F is conjugated to DT or CRM197, 19A is conjugated topneumolysin or TT and the remaining serotypes are split between PD, TT,pneumolysin and optionally PhtD or PhtD/E fusion protein. In a furtherembodiment, 19F is conjugated to DT or CRM197, 19A is conjugated topneumolysin or TT, one further saccharide is conjugated to TT, onefurther saccharide is conjugated to PhtD or PhtD/E and all furthersaccharides are conjugated to PD. In a further embodiment 19F isconjugated to DT or CRM197, 19A is conjugated to pneumolysin, onefurther saccharide is conjugated to TT, one further saccharide isconjugated to pneumolysin, 2 further saccharides are conjugated to PhtDor PhtD/E and all further saccharides are conjugated to PD.

In one embodiment, the immunogenic composition of the inventioncomprises protein D from Haemophilus influenzae. Within this embodiment,If PD is not one of the carrier proteins used to conjugate anysaccharides other than 19F, for example 19F is conjugated to DT whilstthe other serotypes are conjugated to one or more different carrierproteins which are not PD, then PD will be present in the vaccinecomposition as free protein. If PD is one of the carrier proteins usedto conjugate saccharides other than 19F, then PD may optionally bepresent in the vaccine composition as free protein.

The term “saccharide” throughout this specification may indicatepolysaccharide or oligosaccharide and includes both. Polysaccharides areisolated from bacteria and may be sized to some degree by known methods(see for example EP497524 and EP497525) and preferably bymicrofluidisation. Polysaccharides can be sized in order to reduceviscosity in polysaccharide samples and/or to improve filterability forconjugated products. Oligosaccharides have a low number of repeat units(typically 5-30 repeat units) and are typically hydrolysedpolysaccharides

Capsular polysaccharides of Streptococcus pneumoniae comprise repeatingoligosaccharide units which may contain up to 8 sugar residues. For areview of the oligosaccharide units for the key Streptococcus pneumoniaeserotypes see JONES, Christopher. Vaccines based on the cell surfacecarbohydrates of pathogenic bacteria. An. Acad. Bras. Ciénc., June 2005,vol. 77, no. 2, p. 293-324. ISSN 0001-3765. In one embodiment, acapsular saccharide antigen may be a full length polysaccharide, howeverin others it may be one oligosaccharide unit, or a shorter than nativelength saccharide chain of repeating oligosaccharide units. In oneembodiment, all of the saccharides present in the vaccine arepolysaccharides. Full length polysaccharides may be “sized” i.e. theirsize may be reduced by various methods such as acid hydrolysistreatment, hydrogen peroxide treatment, sizing by Emulsiflex® followedby a hydrogen peroxide treatment to generate oligosaccharide fragmentsor microfluidization.

The inventors have also noted that the focus of the art has been to useoligosaccharides for ease of conjugate production. The inventors havefound that by using native or slightly sized polysaccharide conjugates,one or more of the following advantages may be realised: 1) a conjugatehaving high immunogenicity which is filterable, 2) the ratio ofpolysaccharide to protein in the conjugate can be altered such that theratio of polysaccharide to protein (w/w) in the conjugate may beincreased (which can have an effect on the carrier suppression effect),3) immunogenic conjugates prone to hydrolysis may be stabilised by theuse of larger saccharides for conjugation. The use of largerpolysaccharides can result in more cross-linking with the conjugatecarrier and may lessen the liberation of free saccharide from theconjugate. The conjugate vaccines described in the prior art tend todepolymerise the polysaccharides prior to conjugation in order toimprove conjugation. The present inventors have found that saccharideconjugate vaccines retaining a larger size of saccharide can provide agood immune response against pneumococcal disease.

The immunogenic composition of the invention may thus comprise one ormore saccharide conjugates wherein the average size (e.g. weight-averagemolecular weight; M,) of each saccharide before conjugation is above 80kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa or 1000 kDa. In oneembodiment one or more saccharide conjugates of the invention shouldhave an average size of saccharide pre-conjugation of 50-1600, 80-1400,100-1000, 150-500, or 200-400 kDa (note that where average size isM_(w), ‘kDa’ units should be replaced herein with ‘×10³’). In oneembodiment the conjugate post conjugation should be readily filterablethrough a 0.2 micron filter such that a yield of more than 50, 60, 70,80, 90 or 95% is obtained post filtration compared with the prefiltration sample.

For the purposes of the invention, “native polysaccharide” refers to asaccharide that has not been subjected to a process (e.g.post-purification), the purpose of which is to reduce the size of thesaccharide. A polysaccharide can become slightly reduced in size duringnormal purification procedures. Such a saccharide is still native. Onlyif the polysaccharide has been subjected to sizing techniques would thepolysaccharide not be considered native.

For the purposes of the invention, “sized by a factor up to ×2” meansthat the saccharide is subject to a process intended to reduce the sizeof the saccharide but to retain a size more than half the size of thenative polysaccharide. ×3, ×4 etc. are to be interpreted in the same wayi.e. the saccharide is subject to a process intended to reduce the sizeof the polysaccharide but to retain a size more than a third, a quarteretc. the size of the native polysaccharide.

In an aspect of the invention, the immunogenic composition comprisesStreptococcus pneumoniae saccharides from at least 10 serotypesconjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7,8, 9 or each S. pneumoniae saccharide is native polysaccharide.

In an aspect of the invention, the immunogenic composition comprisesStreptococcus pneumoniae saccharides from at least 10 serotypesconjugated to a carrier protein, wherein at least 1, 2, 3, 4, 5, 6, 7,8, 9 or each S. pneumoniae saccharide is sized by a factor up to ×2, ×3,×4, ×5, ×6, ×7, ×8, ×9 or ×10. In one embodiment of this aspect, themajority of the saccharides, for example 6, 7, 8 or more of thesaccharides are sized by a factor up to ×2, ×3, ×4, ×5, ×6, ×7, ×8, ×9or ×10.

The molecular weight or average molecular weight (or size) of asaccharide herein refers to the weight-average molecular weight (Mw) ofthe saccharide measured prior to conjugation and is measured by MALLS.

The MALLS technique is well known in the art and is typically carriedout as described in example 2. For MALLS analysis of pneumococcalsaccharides, two columns (TSKG6000 and 5000PWxl) may be used incombination and the saccharides are eluted in water. Saccharides aredetected using a light scattering detector (for instance Wyatt Dawn DSPequipped with a 10 mW argon laser at 488 nm) and an inferometricrefractometer (for instance Wyatt Otilab DSP equipped with a P100 celland a red filter at 498 nm).

In an embodiment the S. pneumoniae saccharides are nativepolysaccharides or native polysaccharides which have been reduced insize during a normal extraction process.

In an embodiment, the S. pneumoniae saccharides are sized by mechanicalcleavage, for instance by microfluidisation or sonication.Microfluidisation and sonication have the advantage of decreasing thesize of the larger native polysaccharides sufficiently to provide afilterable conjugate. Sizing is by a factor of no more than ×20, ×10,×8, ×6, ×5, ×4, ×3 or ×2.

In an embodiment, the immunogenic composition comprises S. pneumoniaeconjugates that are made from a mixture of native polysaccharides andsaccharides that are sized by a factor of no more than ×20. In oneaspect of this embodiment, the majority of the saccharides, for example6, 7, 8 or more of the saccharides are sized by a factor of up to ×2,×3, ×4, ×5 or ×6.

In an embodiment, the Streptococcus pneumoniae saccharide is conjugatedto the carrier protein via a linker, for instance a bifunctional linker.The linker is optionally heterobifunctional or homobifunctional, havingfor example a reactive amino group and a reactive carboxylic acid group,2 reactive amino groups or two reactive carboxylic acid groups. Thelinker has for example between 4 and 20, 4 and 12, 5 and 10 carbonatoms. A possible linker is ADH. Other linkers include B-propionamido(WO 00/10599), nitrophenyl-ethylamine (Gever et al (1979) Med.Microbiol. Immunol. 165; 171-288), haloalkyl halides (U.S. Pat. No.4,057,685), glycosidic linkages (U.S. Pat. Nos. 4,673,574, 4,808,700),hexane diamine and 6-aminocaproic acid (U.S. Pat. No. 4,459,286). In anembodiment, ADH is used as a linker for conjugating saccharide fromserotype 18C. In an embodiment, ADH is used as a linker for conjugatingsaccharide from serotype 22F.

The saccharide conjugates present in the immunogenic compositions of theinvention may be prepared by any known coupling technique. Theconjugation method may rely on activation of the saccharide with1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form acyanate ester. The activated saccharide may thus be coupled directly orvia a spacer (linker) group to an amino group on the carrier protein.For example, the spacer could be cystamine or cysteamine to give athiolated polysaccharide which could be coupled to the carrier via athioether linkage obtained after reaction with a maleimide-activatedcarrier protein (for example using GMBS) or a haloacetylated carrierprotein (for example using iodoacetimide [e.g. ethyl iodoacetimide HCl]or N-succinimidyl bromoacetate or SIAB, or SIA, or SBAP). Preferably,the cyanate ester (optionally made by CDAP chemistry) is coupled withhexane diamine or ADH and the amino-derivatised saccharide is conjugatedto the carrier protein using carbodiimide (e.g. EDAC or EDC) chemistryvia a carboxyl group on the protein carrier. Such conjugates aredescribed in PCT published application WO 93/15760 Uniformed ServicesUniversity and WO 95/08348 and WO 96/29094

Other suitable techniques use carbodiimides, hydrazides, active esters,norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU.Many are described in WO 98/42721. Conjugation may involve a carbonyllinker which may be formed by reaction of a free hydroxyl group of thesaccharide with CDI (Bethell et al J. Biol. Chem. 1979, 254; 2572-4,Hearn et al J. Chromatogr. 1981. 218; 509-18) followed by reaction ofwith a protein to form a carbamate linkage. This may involve reductionof the anomeric terminus to a primary hydroxyl group, optionalprotection/deprotection of the primary hydroxyl group' reaction of theprimary hydroxyl group with CDI to form a CDI carbamate intermediate andcoupling the CDI carbamate intermediate with an amino group on aprotein.

The conjugates can also be prepared by direct reductive aminationmethods as described in U.S. Pat. No. 4,365,170 (Jennings) and U.S. Pat.No. 4,673,574 (Anderson). Other methods are described in EP-0-161-188,EP-208375 and EP-0-477508.

A further method involves the coupling of a cyanogen bromide (or CDAP)activated saccharide derivatised with adipic acid dihydrazide (ADH) tothe protein carrier by Carbodiimide condensation (Chu C. et al Infect.Immunity, 1983 245 256), for example using EDAC.

In an embodiment, a hydroxyl group (preferably an activated hydroxylgroup for example a hydroxyl group activated to make a cyanate ester[e.g. with CDAP]) on a saccharide is linked to an amino or carboxylicgroup on a protein either directly or indirectly (through a linker).Where a linker is present, a hydroxyl group on a saccharide ispreferably linked to an amino group on a linker, for example by usingCDAP conjugation. A further amino group in the linker for example ADH)may be conjugated to a carboxylic acid group on a protein, for exampleby using carbodiimide chemistry, for example by using EDAC. In anembodiment, the pneumococcal capsular saccharide(s) is conjugated to thelinker first before the linker is conjugated to the carrier protein.Alternatively the linker may be conjugated to the carrier beforeconjugation to the saccharide.

A combination of techniques may also be used, with somesaccharide-protein conjugates being prepared by CDAP, and some byreductive amination.

In general the following types of chemical groups on a protein carriercan be used for coupling/conjugation:

A) Carboxyl (for instance via aspartic acid or glutamic acid). In oneembodiment this group is linked to amino groups on saccharides directlyor to an amino group on a linker with carbodiimide chemistry e.g. withEDAC.

B) Amino group (for instance via lysine). In one embodiment this groupis linked to carboxyl groups on saccharides directly or to a carboxylgroup on a linker with carbodiimide chemistry e.g. with EDAC. In anotherembodiment this group is linked to hydroxyl groups activated with CDAPor CNBr on saccharides directly or to such groups on a linker; tosaccharides or linkers having an aldehyde group; to saccharides orlinkers having a succinimide ester group.

C) Sulphydryl (for instance via cysteine). In one embodiment this groupis linked to a bromo or chloro acetylated saccharide or linker withmaleimide chemistry. In one embodiment this group is activated/modifiedwith bis diazobenzidine.

D) Hydroxyl group (for instance via tyrosine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.

E) Imidazolyl group (for instance via histidine). In one embodiment thisgroup is activated/modified with bis diazobenzidine.

F) Guanidyl group (for instance via arginine).

G) Indolyl group (for instance via tryptophan).

On a saccharide, in general the following groups can be used for acoupling: OH, COOH or NH2. Aldehyde groups can be generated afterdifferent treatments known in the art such as: periodate, acidhydrolysis, hydrogen peroxide, etc.

Direct Coupling Approaches:

Saccharide-OH+CNBr or CDAP----->cyanate ester+NH2-Prot---->conjugate

Saccharide-aldehyde+NH2-Prot---->Schiff base+NaCNBH3---->conjugate

Saccharide-COOH+NH2-Prot+EDAC---->conjugate

Saccharide-NH2+COOH-Prot+EDAC---->conjugate

Indirect coupling via spacer (linker) approaches:

Saccharide-OH+CNBr or CDAP--->cyanateester+NH2----NH2---->saccharide----NH2+COOH-Prot+EDAC----->conjugate

Saccharide-OH+CNBr or CDAP---->cyanateester+NH2-----SH----->saccharide----SH+SH-Prot (native Protein with anexposed cysteine or obtained after modification of amino groups of theprotein by SPDP for instance)----->saccharide-S—S-Prot

Saccharide-OH+CNBr or CDAP--->cyanateester+NH2----SH------->saccharide----SH+maleimide-Prot (modification ofamino groups)---->conjugate

Saccharide-OH+CNBr or CDAP--->cyanateester+NH2-----SH--->Saccharide-SH+haloacetylated-Prot---->Conjugate

Saccharide-COOH+EDAC+NH2-----NH2--->saccharide------NH2+EDAC+COOH-Prot---->conjugate

Saccharide-COOH+EDAC+NH2----SH----->saccharide----SH+SH-Prot (nativeProtein with an exposed cysteine or obtained after modification of aminogroups of the protein by SPDP for instance)----->saccharide-S—S-Prot

Saccharide-COOH+EDAC+NH2----SH----->saccharide----SH+maleimide-Prot(modification of amino groups)---->conjugate

Saccharide-COOH+EDAC+NH2----SH--->Saccharide-SH+haloacetylated-Prot---->Conjugate

Saccharide-Aldehyde+NH2-----NH2---->saccharide---NH2+EDAC+COOH-Prot---->conjugate

Note: instead of EDAC above, any suitable carbodiimide may be used.

In summary, the types of protein carrier chemical group that may begenerally used for coupling with a saccharide are amino groups (forinstance on lysine residues), COOH groups (for instance on aspartic andglutamic acid residues) and SH groups (if accessible) (for instance oncysteine residues.

Preferably the ratio of carrier protein to S. pneumoniae saccharide isbetween 1:5 and 5:1; e.g. between 1:0.5-4:1, 1:1-3.5:1, 1.2:1-3:1,1.5:1-2.5:1; e.g. between 1:2 and 2.5:1; 1:1 and 2:1 (w/w). In anembodiment, the majority of the conjugates, for example 6, 7, 8, 9 ormore of the conjugates have a ratio of carrier protein to saccharidethat is greater than 1:1, for example 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1or 1.6:1.

In an embodiment, at least one S. pneumoniae saccharide is conjugated toa carrier protein via a linker using CDAP and EDAC. For example, 18C or22F may be conjugated to a protein via a linker (for example those withtwo hydrazino groups at its ends such as ADH) using CDAP and EDAC asdescribed above. When a linker is used, CDAP may be used to conjugatethe saccharide to a linker and EDAC may then be used to conjugate thelinker to a protein or, alternatively EDAC may be used first toconjugate the linker to the protein, after which CDAP may be used toconjugate the linker to the saccharide.

In general, the immunogenic composition of the invention may comprise adose of each saccharide conjugate between 0.1 and 20 μg, 1 and 10 μg or1 and 3 μg of saccharide.

In an embodiment, the immunogenic composition of the invention containseach S. pneumoniae capsular saccharide at a dose of between 0.1-20 μg;0.5-10 μg; 0.5-5 μg or 1-3 μg of saccharide. In an embodiment, capsularsaccharides may be present at different dosages, for example somecapsular saccharides may be present at a dose of exactly 1 μg or somecapsular saccharides may be present at a dose of exactly 3 μg. In anembodiment, saccharides from serotypes 3, 18C and 19F (or 4, 18C and19F) are present at a higher dose than other saccharides. In one aspectof this embodiment, serotypes 3, 18C and 19F (or 4, 18C and 19F) arepresent at a dose of around or exactly 3 μg whilst other saccharides inthe immunogenic composition are present at a dose of around or exactly 1μg.

“Around” or “approximately” are defined as within 10% more or less ofthe given figure for the purposes of the invention.

In an embodiment, at least one of the S. pneumoniae capsular saccharidesis directly conjugated to a carrier protein (e.g. using one of thechemistries described above); Preferably the at least one of the S.pneumoniae capsular saccharides is directly conjugated by CDAP. In anembodiment, the majority of the capsular saccharides for example 5, 6,7, 8, 9 or more are directly linked to the carrier protein by CDAP (seeWO 95/08348 and WO 96/29094).

The immunogenic composition may comprise Streptococcus pneumoniaeproteins, herein termed Streptococcus pneumoniae proteins of theinvention. Such proteins may be used as carrier proteins, or may bepresent as free proteins, or may be present both as carrier proteins andas free proteins. The Streptococcus pneumoniae proteins of the inventionare either surface exposed, at least during part of the life cycle ofthe pneumococcus, or are proteins which are secreted or released by thepneumococcus. Preferably the proteins of the invention are selected fromthe following categories, such as proteins having a Type II Signalsequence motif of LXXC (where X is any amino acid, e.g., thepolyhistidine triad family (PhtX)), choline binding proteins (CbpX),proteins having a Type I Signal sequence motif (e.g., Sp101), proteinshaving a LPXTG motif (where X is any amino acid, e.g., Sp128, Sp130),and toxins (e.g., Ply). Preferred examples within these categories (ormotifs) are the following proteins, or immunologically functionalequivalents thereof.

In one embodiment, the immunogenic composition of the inventioncomprises at least 1 protein selected from the group consisting of thePoly Histidine Triad family (PhtX), Choline Binding Protein family(CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytXtruncate chimeric proteins (or fusions), pneumolysin (Ply), PspA, PsaA,Sp128, Sp101, Sp130, Sp125 and Sp133. In a further embodiment, theimmunogenic composition comprises 2 or more proteins selected from thegroup consisting of the Poly Histidine Triad family (PhtX), CholineBinding Protein family (CbpX), CbpX truncates, LytX family, LytXtruncates, CbpX truncate-LytX truncate chimeric proteins (or fusions),pneumolysin (Ply), PspA, PsaA, and Sp128. In one more embodiment, theimmunogenic composition comprises 2 or more proteins selected from thegroup consisting of the Poly Histidine Triad family (PhtX), CholineBinding Protein family (CbpX), CbpX truncates, LytX family, LytXtruncates, CbpX truncate-LytX truncate chimeric proteins (or fusions),pneumolysin (Ply), and Sp128.

The Pht (Poly Histidine Triad) family comprises proteins PhtA, PhtB,PhtD, and PhtE. The family is characterized by a lipidation sequence,two domains separated by a proline-rich region and several histidinetriads, possibly involved in metal or nucleoside binding or enzymaticactivity, (3-5) coiled-coil regions, a conserved N-terminus and aheterogeneous C terminus. It is present in all strains of pneumococcitested. Homologous proteins have also been found in other Streptococciand Neisseria. In one embodiment of the invention, the Pht protein ofthe invention is PhtD. It is understood, however, that the terms Pht A,B, D, and E refer to proteins having sequences disclosed in thecitations below as well as naturally-occurring (and man-made) variantsthereof that have a sequence homology that is at least 90% identical tothe referenced proteins. Preferably it is at least 95% identical andmost preferably it is 97% identical.

With regards to the PhtX proteins, PhtA is disclosed in WO 98/18930, andis also referred to Sp36. As noted above, it is a protein from thepolyhistidine triad family and has the type II signal motif of LXXC.PhtD is disclosed in WO 00/37105, and is also referred to Sp036D. Asnoted above, it also is a protein from the polyhistidine triad familyand has the type II LXXC signal motif. PhtB is disclosed in WO 00/37105,and is also referred to Sp036B. Another member of the PhtB family is theC3-Degrading Polypeptide, as disclosed in WO 00/17370. This protein alsois from the polyhistidine triad family and has the type II LXXC signalmotif. A preferred immunologically functional equivalent is the proteinSp42 disclosed in WO 98/18930. A PhtB truncate (approximately 79 kD) isdisclosed in WO99/15675 which is also considered a member of the PhtXfamily. PhtE is disclosed in WO00/30299 and is referred to as BVH-3.Where any Pht protein is referred to herein, it is meant thatimmunogenic fragments or fusions thereof of the Pht protein can be used.For example, a reference to PhtX includes immunogenic fragments orfusions thereof from any Pht protein. A reference to PhtD or PhtB isalso a reference to PhtDE or PhtBE fusions as found, for example, inWO0198334.

Pneumolysin is a multifunctional toxin with a distinct cytolytic(hemolytic) and complement activation activities (Rubins et al., Am.Respi. Cit Care Med, 153:1339-1346 (1996)). The toxin is not secreted bypneumococci, but it is released upon lysis of pneumococci under theinfluence of autolysin. Its effects include e.g., the stimulation of theproduction of inflammatory cytokines by human monocytes, the inhibitionof the beating of cilia on human respiratory epithelial, and thedecrease of bactericidal activity and migration of neutrophils. The mostobvious effect of pneumolysin is in the lysis of red blood cells, whichinvolves binding to cholesterol. Because it is a toxin, it needs to bedetoxified (i.e., non-toxic to a human when provided at a dosagesuitable for protection) before it can be administered in vivo.Expression and cloning of wild-type or native pneumolysin is known inthe art. See, for example, Walker et al. (Infect Immun, 55:1184-1189(1987)), Mitchell et al. (Biochim Biophys Acta, 1007:67-72 (1989) andMitchell et al (NAR, 18:4010 (1990)). Detoxification of ply can beconducted by chemical means, e.g., subject to formalin or glutaraldehydetreatment or a combination of both (WO 04081515, PCT/EP2005/010258).Such methods are well known in the art for various toxins.Alternatively, ply can be genetically detoxified. Thus, the inventionencompasses derivatives of pneumococcal proteins which may be, forexample, mutated proteins. The term “mutated” is used herein to mean amolecule which has undergone deletion, addition or substitution of oneor more amino acids using well known techniques for site directedmutagenesis or any other conventional method. For example, as describedabove, a mutant ply protein may be altered so that it is biologicallyinactive whilst still maintaining its immunogenic epitopes, see, forexample, WO90/06951, Berry et al. (Infect Immun, 67:981-985 (1999)) andWO99/03884.

As used herein, it is understood that the term “Ply” refers to mutatedor detoxified pneumolysin suitable for medical use (i.e., non toxic).

Concerning the Choline Binding Protein family (CbpX), members of thatfamily were originally identified as pneumococcal proteins that could bepurified by choline-affinity chromatography. All of the choline-bindingproteins are non-covalently bound to phosphorylcholine moieties of cellwall teichoic acid and membrane-associated lipoteichoic acid.Structurally, they have several regions in common over the entirefamily, although the exact nature of the proteins (amino acid sequence,length, etc.) can vary. In general, choline binding proteins comprise anN terminal region (N), conserved repeat regions (R1 and/or R2), aproline rich region (P) and a conserved choline binding region (C), madeup of multiple repeats, that comprises approximately one half of theprotein. As used in this application, the term “Choline Binding Proteinfamily (CbpX)” is selected from the group consisting of Choline BindingProteins as identified in WO97/41151, PbcA, SpsA, PspC, CbpA, CbpD, andCbpG. CbpA is disclosed in WO97/41151. CbpD and CbpG are disclosed inWO00/29434. PspC is disclosed in WO97/09994. PbcA is disclosed inWO98/21337. SpsA is a Choline binding protein disclosed in WO 98/39450.Preferably the Choline Binding Proteins are selected from the groupconsisting of CbpA, PbcA, SpsA and PspC.

Another preferred embodiment is CbpX truncates wherein “CbpX” is definedabove and “truncates” refers to CbpX proteins lacking 50% or more of theCholine binding region (C). Preferably such proteins lack the entirecholine binding region. More preferably, the such protein truncates lack(i) the choline binding region and (ii) a portion of the N-terminal halfof the protein as well, yet retain at least one repeat region (R1 orR2). More preferably still, the truncate has 2 repeat regions (R1 andR2). Examples of such preferred embodiments are NR1xR2 and R1xR2 asillustrated in WO99/51266 or WO99/51188, however, other choline bindingproteins lacking a similar choline binding region are also contemplatedwithin the scope of this invention.

The LytX family is membrane associated proteins associated with celllysis. The N-terminal domain comprises choline binding domain(s),however the LytX family does not have all the features found in the CbpAfamily noted above and thus for the present invention, the LytX familyis considered distinct from the CbpX family. In contrast with the CbpXfamily, the C-terminal domain contains the catalytic domain of the LytXprotein family. The family comprises LytA, B and C. With regards to theLytX family, LytA is disclosed in Ronda et al., Eur J Biochem,164:621-624 (1987). LytB is disclosed in WO 98/18930, and is alsoreferred to as Sp46. LytC is also disclosed in WO 98/18930, and is alsoreferred to as Sp91. A preferred member of that family is LytC.

Another preferred embodiment are LytX truncates wherein “LytX” isdefined above and “truncates” refers to LytX proteins lacking 50% ormore of the Choline binding region. Preferably such proteins lack theentire choline binding region. Yet another preferred embodiment of thisinvention are CbpX truncate-LytX truncate chimeric proteins (orfusions). Preferably this comprises NR1xR2 (or R1xR2) of CbpX and theC-terminal portion (Cterm, i.e., lacking the choline binding domains) ofLytX (e.g., LytCCterm or Sp91Cterm). More preferably CbpX is selectedfrom the group consisting of CbpA, PbcA, SpsA and PspC. More preferablystill, it is CbpA. Preferably, LytX is LytC (also referred to as Sp91).Another embodiment of the present invention is a PspA or PsaA truncateslacking the choline binding domain (C) and expressed as a fusion proteinwith LytX. Preferably, LytX is LytC.

With regards to PsaA and PspA, both are know in the art. For example,PsaA and transmembrane deletion variants thereof have been described byBerry & Paton, Infect Immun 1996 December; 64(12):5255-62. PspA andtransmembrane deletion variants thereof have been disclosed in, forexample, U.S. Pat. No. 5,804,193, WO 92/14488, and WO 99/53940.

Sp128 and Sp130 are disclosed in WO00/76540. Sp125 is an example of apneumococcal surface protein with the Cell Wall Anchored motif of LPXTG(where X is any amino acid). Any protein within this class ofpneumococcal surface protein with this motif has been found to be usefulwithin the context of this invention, and is therefore considered afurther protein of the invention. Sp125 itself is disclosed in WO98/18930, and is also known as ZmpB—a zinc metalloproteinase. Sp101 isdisclosed in WO 98/06734 (where it has the reference #y85993). It ischaracterized by a Type I signal sequence. Sp133 is disclosed in WO98/06734 (where it has the reference #y85992). It is also characterizedby a Type I signal sequence.

Examples of preferred Moraxella catarrhalis protein antigens which canbe included in a combination vaccine (especially for the prevention ofotitis media) are: OMP106 [WO 97/41731 (Antex) & WO 96/34960 (PMC)];OMP21 or fragments thereof (WO 0018910); LbpA &/or LbpB [WO 98/55606(PMC)]; TbpA &/or TbpB [WO 97/13785 & WO 97/32980 (PMC)]; CopB [HelminenM E, et al. (1993) Infect. Immun. 61:2003-2010]; UspA1 and/or UspA2 [WO93/03761 (University of Texas)]; OmpCD; HasR (PCT/EP99/03824); PiIQ(PCT/EP99/03823); OMP85 (PCT/EP00/01468); lipo06 (GB 9917977.2); lipo10(GB 9918208.1); lipo11 (GB 9918302.2); lipo18 (GB 9918038.2); P6(PCT/EP99/03038); D15 (PCT/EP99/03822); OmplA1 (PCT/EP99/06781); Hly3(PCT/EP99/03257); and OmpE. Examples of non-typeable Haemophilusinfluenzae antigens or fragments thereof which can be included in acombination vaccine (especially for the prevention of otitis media)include: Fimbrin protein [(U.S. Pat. No. 5,766,608—Ohio State ResearchFoundation)] and fusions comprising peptides therefrom [eg LB1(f)peptide fusions; U.S. Pat. No. 5,843,464 (OSU) or WO 99/64067]; OMP26[WO 97/01638 (Cortecs)]; P6 [EP 281673 (State University of New York)];TbpA and/or TbpB; Hia; Hsf; Hin47; Hif; Hmw1; Hmw2; Hmw3; Hmw4; Hap; D15(WO 94/12641); P2; and P5 (WO 94/26304).

The proteins of the invention may also be beneficially combined. Bycombined is meant that the immunogenic composition comprises all of theproteins from within the following combinations, either as carrierproteins or as free proteins or a mixture of the two. For example, in acombination of two proteins as set out hereinafter, both proteins may beused as carrier proteins, or both proteins may be present as freeproteins, or both may be present as carrier and as free protein, or onemay be present as a carrier protein and a free protein whilst the otheris present only as a carrier protein or only as a free protein, or onemay be present as a carrier protein and the other as a free protein.Where a combination of three proteins is given, similar possibilitiesexist. Preferred combinations include, but are not limited to,PhtD+NR1xR2, PhtD+NR1xR2-Sp91Cterm chimeric or fusion proteins,PhtD+Ply, PhtD+Sp128, PhtD+PsaA, PhtD+PspA, PhtA+NR1xR2,PhtA+NR1xR2-Sp91Cterm chimeric or fusion proteins, PhtA+Ply, PhtA+Sp128,PhtA+PsaA, PhtA+PspA, NR1xR2+LytC, NR1xR2+PspA, NR1xR2+PsaA,NR1xR2+Sp128, R1xR2+LytC, R1xR2+PspA, R1xR2+PsaA, R1xR2+Sp128,R1xR2+PhtD, R1xR2+PhtA. Preferably, NR1xR2 (or R1xR2) is from CbpA orPspC. More preferably it is from CbpA. Other combinations include 3protein combinations such as PhtD+NR1xR2+Ply, and PhtA+NR1xR2+PhtD. Inone embodiment, the vaccine composition comprises detoxified pneumolysinand PhtD or PhtDE as carrier proteins. In a further embodiment, thevaccine composition comprises detoxified pneumolysin and PhtD or PhtDEas free proteins.

In an independent aspect, the present invention provides an immunogeniccomposition comprising at least four S. pneumoniae capsular saccharideconjugates containing saccharides from different S. pneumoniae serotypeswherein at least one saccharide is conjugated to PhtD or fusion proteinthereof and the immunogenic composition is capable of eliciting aneffective immune response against PhtD.

An effective immune response against PhtD or fusion protein thereof ismeasured for example by a protection assay such as that described inexample 15. An effective immune response provides at least 40%, 50%,60%, 70%, 80% or 90% survival 7 days after challenge with a heterologousstrain. Given that the challenge strain is heterologous, the protectionafforded is due to the immune response against PhtD or fusion proteinthereof.

Alternatively, an effective immune response against PhtD is measured byELISA as described in example 14. An effective immune response gives ananti-PhtD IgG response of at least 250, 300, 350, 400, 500, 550 or 600μg/ml GMC.

For example, the immunogenic composition comprises at least 2, 3, 4, 5,6, 7, 8, 9 or 10 S. pneumoniae capsular saccharides from differentserotypes conjugated to PhtD or fusion protein thereof. For exampleserotypes 22F and 1, 2, 3, 4, 5, 6 or 7 further selected from serotypes1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C,19A, 19F, 20, 23F and 33F are conjugated to PhtD. In an embodiment twoor three of serotypes 3, 6A and 22F are conjugated to PhtD or fusionprotein thereof.

In an embodiment, the immunogenic composition of the invention comprisesat least one S. pneumoniae capsular saccharide conjugated to PhtD orfusion protein thereof via a linker, for example ADH. In an embodiment,one of the conjugation chemistries listed below is used.

In an embodiment, the immunogenic composition of the invention comprisesat least one S. pneumoniae capsular saccharide conjugated to PhtD orfusion protein thereof, wherein the ratio of PhtD to saccharide in theconjugate is between 6:1 and 1:5, 6:1 and 2:1, 6:1 and 2.5:1, 6:1 and3:1, 6:1 and 3.5:1 (w/w) or is greater than (i.e. contains a largerproportion of PhtD) 2.0:1, 2.5:1, 3.0:1, 3.5:1 or 4.0:1 (w/w).

In an embodiment, the immunogenic composition of the invention comprisespneumolysin.

The present invention further provides a vaccine containing theimmunogenic compositions of the invention and a pharmaceuticallyacceptable excipient.

The vaccines of the present invention may be adjuvanted, particularlywhen intended for use in an elderly population but also for use ininfant populations. Suitable adjuvants include an aluminum salt such asaluminum hydroxide gel or aluminum phosphate or alum, but may also beother metal salts such as those of calcium, magnesium, iron or zinc, ormay be an insoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatized saccharides, orpolyphosphazenes.

It is preferred that the adjuvant be selected to be a preferentialinducer of a TH1 type of response. Such high levels of Th1-typecytokines tend to favour the induction of cell mediated immune responsesto a given antigen, whilst high levels of Th2-type cytokines tend tofavour the induction of humoral immune responses to the antigen.

The distinction of Th1 and Th2-type immune response is not absolute. Inreality an individual will support an immune response which is describedas being predominantly Th1 or predominantly Th2. However, it is oftenconvenient to consider the families of cytokines in terms of thatdescribed in murine CD4+ve T cell clones by Mosmann and Coffman(Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. (Annual Review of Immunology, 7, p₁₄₅-173). Traditionally,Th1-type responses are associated with the production of the INF-γ andIL-2 cytokines by T-lymphocytes. Other cytokines often directlyassociated with the induction of Th1-type immune responses are notproduced by T-cells, such as IL-12. In contrast, Th2-type responses areassociated with the secretion of 11-4, IL-5, IL-6, IL-10. Suitableadjuvant systems which promote a predominantly Th1 response include:Monophosphoryl lipid A or a derivative thereof (or detoxified lipid A ingeneral—see for instance WO2005107798), particularly 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL) (for its preparation see GB 2220211 A);and a combination of monophosphoryl lipid A, preferably 3-de-O-acylatedmonophosphoryl lipid A, together with either an aluminum salt (forinstance aluminum phosphate or aluminum hydroxide) or an oil-in-wateremulsion. In such combinations, antigen and 3D-MPL are contained in thesame particulate structures, allowing for more efficient delivery ofantigenic and immunostimulatory signals. Studies have shown that 3D-MPLis able to further enhance the immunogenicity of an alum-adsorbedantigen [Thoelen et al. Vaccine (1998) 16:708-14; EP 689454-B1].

An enhanced system involves the combination of a monophosphoryl lipid Aand a saponin derivative, particularly the combination of QS21 and3D-MPL as disclosed in WO 94/00153, or a less reactogenic compositionwhere the QS21 is quenched with cholesterol as disclosed in WO 96/33739.A particularly potent adjuvant formulation involving QS21, 3D-MPL andtocopherol in an oil in water emulsion is described in WO 95/17210. Inone embodiment the immunogenic composition additionally comprises asaponin, which may be QS21. The formulation may also comprise an oil inwater emulsion and tocopherol (WO 95/17210). Unmethylated CpG containingoligonucleotides (WO 96/02555) and other immunomodulatoryoligonucleotides (WO0226757 and WO03507822) are also preferentialinducers of a TH1 response and are suitable for use in the presentinvention.

Particular adjuvants are those selected from the group of metal Salts,oil in water emulsions, Toll like receptors agonist, (in particular Tolllike receptor 2 agonist, Toll like receptor 3 agonist, Toll likereceptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8agonist and Toll like receptor 9 agonist), saponins or combinationsthereof.

An adjuvant that can be used with the vaccine compositions of theinvention are bleb or outer membrane vesicle preparations from Gramnegative bacterial strains such as those taught byWO02/09746—particularly N. meningitidis blebs. Adjuvant properties ofblebs can be improved by retaining LOS (lipooligosacccharide) on itssurface (e.g. through extraction with low concentrations of detergent[for instanct 0-0.1% deoxycholate]). LOS can be detoxified through themsbB(−) or htrB(−) mutations discussed in WO02/09746. Adjuvantproperties can also be improved by retaining PorB (and optionallyremoving PorA) from meningococcal blebs. Adjuvant properties can also beimproved by truncating the outer core saccharide structure of LOS onmeningococcal blebs—for instance via the IgtB(−) mutation discussed inWO2004/014417. Alternatively, the aforementioned LOS (e.g. isolated froma msbB(−) and/or IgtB(−) strain) can be purified and used as an adjuvantin the compositions of the invention.

A further adjuvant which may be used with the compositions of theinvention may be selected from the group: a saponin, lipid A or aderivative thereof, an immunostimulatory oligonucleotide, an alkylglucosaminide phosphate, an oil in water emulsion or combinationsthereof. A further preferred adjuvant is a metal salt in combinationwith another adjuvant. It is preferred that the adjuvant is a Toll likereceptor agonist in particular an agonist of a Toll like receptor 2, 3,4, 7, 8 or 9, or a saponin, in particular Qs21. It is further preferredthat the adjuvant system comprises two or more adjuvants from the abovelist. In particular the combinations preferably contain a saponin (inparticular Qs21) adjuvant and/or a Toll like receptor 9 agonist such asa CpG containing immunostimulatory oligonucleotide. Other preferredcombinations comprise a saponin (in particular QS21) and a Toll likereceptor 4 agonist such as monophosphoryl lipid A or its 3 deacylatedderivative, 3 D-MPL, or a saponin (in particular QS21) and a Toll likereceptor 4 ligand such as an alkyl glucosaminide phosphate.

Particularly preferred adjuvants are combinations of 3D-MPL and QS21 (EP0 671 948 B1), oil in water emulsions comprising 3D-MPL and QS21 (WO95/17210, WO 98/56414), or 3D-MPL formulated with other carriers (EP 0689 454 B1). Other preferred adjuvant systems comprise a combination of3 D MPL, QS21 and a CpG oligonucleotide as described in U.S. Pat. Nos.6,558,670, 6,544,518.

In an embodiment the adjuvant is (or comprises) a Toll like receptor(TLR) 4 ligand, preferably an agonist such as a lipid A derivativeparticularly monophosphoryl lipid A or more particularly 3 Deacylatedmonophoshoryl lipid A (3 D-MPL). 3 D-MPL is available fromGlaxoSmithKline Biologicals North America and primarily promotes CD4+ Tcell responses with an IFN-g (Th1) phenotype. It can be producedaccording to the methods disclosed in GB 2 220 211 A. Chemically it is amixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6acylated chains. Preferably in the compositions of the present inventionsmall particle 3 D-MPL is used. Small particle 3 D-MPL has a particlesize such that it may be sterile-filtered through a 0.22 m filter. Suchpreparations are described in International Patent Application No. WO94/21292.

Synthetic derivatives of lipid A are known and thought to be TLR 4agonists including, but not limited to:

OM174(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o-phosphono-β-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranosyldihydrogenphosphate),(WO 95/14026)

OM 294 DP (3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1,10-bis(dihydrogenophosphate)(WO99/64301 and WO 00/0462)

OM 197 MP-Ac DP (3S-,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1-dihydrogenophosphate10-(6-aminohexanoate) (WO 01/46127)

Other TLR4 ligands which may be used are alkyl Glucosaminide phosphates(AGPs) such as those disclosed in WO9850399 or U.S. Pat. No. 6,303,347(processes for preparation of AGPs are also disclosed), orpharmaceutically acceptable salts of AGPs as disclosed in U.S. Pat. No.6,764,840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists.Both are thought to be useful as adjuvants.

Another prefered immunostimulant for use in the present invention isQuil A and its derivatives. Quil A is a saponin preparation isolatedfrom the South American tree Quilaja Saponaria Molina and was firstdescribed as having adjuvant activity by Dalsgaard et al. in 1974(“Saponin adjuvants”, Archiv. fur die gesamte Virusforschung, Vol. 44,Springer Verlag, Berlin, p₂₄₃-254). Purified fragments of Quil A havebeen isolated by HPLC which retain adjuvant activity without thetoxicity associated with Quil A (EP 0 362 278), for example QS7 and QS21(also known as QA7 and QA21). QS-21 is a natural saponin derived fromthe bark of Quillaja saponaria Molina which induces CD8+ cytotoxic Tcells (CTLs), Th1 cells and a predominant IgG2a antibody response and isa preferred saponin in the context of the present invention.

Particular formulations of QS21 have been described which areparticularly preferred, these formulations further comprise a sterol(WO96/33739). The saponins forming part of the present invention may beseparate in the form of micelles, mixed micelles (preferentially, butnot exclusively with bile salts) or may be in the form of ISCOM matrices(EP 0 109 942 B1), liposomes or related colloidal structures such asworm-like or ring-like multimeric complexes or lipidic/layeredstructures and lamellae when formulated with cholesterol and lipid, orin the form of an oil in water emulsion (for example as in WO 95/17210).The saponins may preferably be associated with a metallic salt, such asaluminium hydroxide or aluminium phosphate (WO 98/15287).

Preferably, the saponin is presented in the form of a liposome, ISCOM oran oil in water emulsion.

An enhanced system involves the combination of a monophosphoryl lipid A(or detoxified lipid A) and a saponin derivative, particularly thecombination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a lessreactogenic composition where the QS21 is quenched with cholesterol asdisclosed in WO 96/33739. A particularly potent adjuvant formulationinvolving tocopherol with or without QS21 and/or 3D-MPL in an oil inwater emulsion is described in WO 95/17210. In one embodiment theimmunogenic composition additionally comprises a saponin, which may beQS21.

Immunostimulatory oligonucleotides or any other Toll-like receptor (TLR)9 agonist may also be used. The preferred oligonucleotides for use inadjuvants or vaccines of the present invention are CpG containingoligonucleotides, preferably containing two or more dinucleotide CpGmotifs separated by at least three, more preferably at least six or morenucleotides. A CpG motif is a Cytosine nucleotide followed by a Guaninenucleotide. The CpG oligonucleotides of the present invention aretypically deoxynucleotides. In a preferred embodiment theinternucleotide in the oligonucleotide is phosphorodithioate, or morepreferably a phosphorothioate bond, although phosphodiester and otherinternucleotide bonds are within the scope of the invention. Alsoincluded within the scope of the invention are oligonucleotides withmixed internucleotide linkages. Methods for producing phosphorothioateoligonucleotides or phosphorodithioate are described in U.S. Pat. Nos.5,666,153, 5,278,302 and WO95/26204.

Examples of preferred oligonucleotides have the following sequences. Thesequences preferably contain phosphorothioate modified internucleotidelinkages.

OLIGO 1(SEQ ID NO: 1): TCC ATG ACG TTC CTG ACG TT (CpG 1826)OLIGO 2(SEQ ID NO: 2): TCT CCC AGC GTG CCC CAT (CpG 1758)OLIGO 3(SEQ ID NO: 3): ACC GAT GAC GTC GCC GGT GAC GGC ACC ACGOLIGO 4(SEQ ID NO: 4): TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006)OLIGO 5(SEQ ID NO: 5): TCC ATG ACG TTC CTG ATG CT (CpG 1668)OLIGO 6(SEQ ID NO: 6): TCG ACG TTT TCG GCG CGC GCC G (CpG 5456)

Alternative CpG oligonucleotides may comprise the preferred sequencesabove in that they have inconsequential deletions or additions thereto.

The CpG oligonucleotides utilised in the present invention may besynthesized by any method known in the art (for example see EP 468520).Conveniently, such oligonucleotides may be synthesized utilising anautomated synthesizer.

The adjuvant may be an oil in water emulsion or may comprise an oil inwater emulsion in combination with other adjuvants. The oil phase of theemulsion system preferably comprises a metabolisable oil. The meaning ofthe term metabolisable oil is well known in the art. Metabolisable canbe defined as “being capable of being transformed by metabolism”(Dorland's Illustrated Medical Dictionary, W. B. Sanders Company,25^(th) edition (1974)). The oil may be any vegetable oil, fish, oil,animal or synthetic oil, which is not toxic to the recipient and iscapable of being transformed by metabolism. Nuts, seeds, and grains arecommon sources of vegetable oils. Synthetic oils are also part of thisinvention and can include commercially available oils such as NEOBEE®and others. Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oilwhich is found in large quantities in shark-liver oil, and in lowerquantities in olive oil, wheat germ oil, rice bran oil, and yeast, andis a particularly preferred oil for use in this invention. Squalene is ametabolisable oil by virtue of the fact that it is an intermediate inthe biosynthesis of cholesterol (Merck index, 10^(th) Edition, entry no.8619).

Tocols (e.g. vitamin E) are also often used in oil emulsions adjuvants(EP 0 382 271 B1; U.S. Pat. No. 5,667,784; WO 95/17210). Tocols used inthe oil emulsions (preferably oil in water emulsions) of the inventionmay be formulated as described in EP 0 382 271 B1, in that the tocolsmay be dispersions of tocol droplets, optionally comprising anemulsifier, of preferably less than 1 micron in diameter. Alternatively,the tocols may be used in combination with another oil, to form the oilphase of an oil emulsion. Examples of oil emulsions which may be used incombination with the tocol are described herein, such as themetabolisable oils described above.

Oil in water emulsion adjuvants per se have been suggested to be usefulas adjuvant compositions (EP 0 399 843B), also combinations of oil inwater emulsions and other active agents have been described as adjuvantsfor vaccines (WO 95/17210; WO 98/56414; WO 99/12565; WO 99/11241). Otheroil emulsion adjuvants have been described, such as water in oilemulsions (U.S. Pat. No. 5,422,109; EP 0 480 982 B2) and water in oil inwater emulsions (U.S. Pat. No. 5,424,067; EP 0 480 981 B). All of whichform preferred oil emulsion systems (in particular when incorporatingtocols) to form adjuvants and compositions of the present invention.

Most preferably the oil emulsion (for instance oil in water emulsions)further comprises an emulsifier such as TWEEN 80 and/or a sterol such ascholesterol.

A preferred oil emulsion (preferably oil-in-water emulsion) comprises ametabolisible, non-toxic oil, such as squalane, squalene or a tocopherolsuch as alpha tocopherol (and preferably both squalene and alphatocopherol) and optionally an emulsifier (or surfactant) such as Tween80. A sterol (preferably cholesterol) may also be included.

The method of producing oil in water emulsions is well known to the manskilled in the art. Commonly, the method comprises mixing thetocol-containing oil phase with a surfactant such as a PBS/TWEEN80™solution, followed by homogenisation using a homogenizer, it would beclear to a man skilled in the art that a method comprising passing themixture twice through a syringe needle would be suitable forhomogenising small volumes of liquid. Equally, the emulsificationprocess in microfluidiser (M110S Microfluidics machine, maximum of 50passes, for a period of 2 minutes at maximum pressure input of 6 bar(output pressure of about 850 bar)) could be adapted by the man skilledin the art to produce smaller or larger volumes of emulsion. Theadaptation could be achieved by routine experimentation comprising themeasurement of the resultant emulsion until a preparation was achievedwith oil droplets of the required diameter.

In an oil in water emulsion, the oil and emulsifier should be in anaqueous carrier. The aqueous carrier may be, for example, phosphatebuffered saline.

The size of the oil droplets found within the stable oil in wateremulsion are preferably less than 1 micron, may be in the range ofsubstantially 30-600 nm, preferably substantially around 30-500 nm indiameter, and most preferably substantially 150-500 nm in diameter, andin particular about 150 nm in diameter as measured by photon correlationspectroscopy. In this regard, 80% of the oil droplets by number shouldbe within the preferred ranges, more preferably more than 90% and mostpreferably more than 95% of the oil droplets by number are within thedefined size ranges. The amounts of the components present in the oilemulsions of the present invention are conventionally in the range offrom 0.5-20% or 2 to 10% oil (of the total dose volume), such assqualene; and when present, from 2 to 10% alpha tocopherol; and from 0.3to 3% surfactant, such as polyoxyethylene sorbitan monooleate.Preferably the ratio of oil (preferably squalene): tocol (preferablyα-tocopherol) is equal or less than 1 as this provides a more stableemulsion. An emulsifier, such as Tween80 or Span 85 may also be presentat a level of about 1%. In some cases it may be advantageous that thevaccines of the present invention will further contain a stabiliser.

Examples of preferred emulsion systems are described in WO 95/17210, WO99/11241 and WO 99/12565 which disclose emulsion adjuvants based onsqualene, α-tocopherol, and TWEEN 80, optionally formulated with theimmunostimulants QS21 and/or 3D-MPL. Thus in a particularly, preferredembodiment of the present invention, the adjuvant of the invention mayadditionally comprise further immunostimulants, such as LPS orderivatives thereof, and/or saponins. Examples of furtherimmunostimulants are described herein and in “Vaccine Design—The Subunitand Adjuvant Approach” 1995, Pharmaceutical Biotechnology, Volume 6,Eds. Powell, M. F., and Newman, M. J., Plenum Press, New York andLondon, ISBN 0-306-44867-X.

In a preferred aspect the adjuvant and immunogenic compositionsaccording to the invention comprise a saponin (preferably QS21) and/oran LPS derivative (preferably 3D-MPL) in an oil emulsion describedabove, optionally with a sterol (preferably cholesterol). Additionallythe oil emulsion (preferably oil in water emulsion) may contain span 85and/or lecithin and/or tricaprylin. Adjuvants comprising an oil-in-wateremulsion, a sterol and a saponin are described in WO 99/12565.

Typically for human administration the saponin (preferably QS21) and/orLPS derivative (preferably 3D-MPL) will be present in a human dose ofimmunogenic composition in the range of 1 μg-200 μg, such as 10-100 μg,preferably 10 μg-50 μg per dose. Typically the oil emulsion (preferablyoil in water emulsion) will comprise from 2 to 10% metabolisible oil.Preferably it will comprise from 2 to 10% squalene, from 2 to 10% alphatocopherol and from 0.3 to 3% (preferably 0.4-2%) emulsifier (preferablytween 80 [polyoxyethylene sorbitan monooleate]). Where both squalene andalpha tocopherol are present, preferably the ratio of squalene: alphatocopherol is equal to or less than 1 as this provides a more stableemulsion. Span 85 (Sorbitan trioleate) may also be present at a level of0.5 to 1% in the emulsions used in the invention. In some cases it maybe advantageous that the immunogenic compositions and vaccines of thepresent invention will further contain a stabiliser, for example otheremulsifiers/surfactants, including caprylic acid (merck index 10^(th)Edition, entry no. 1739), of which Tricaprylin is particularlypreferred.

Where squalene and a saponin (preferably QS21) are included, it is ofbenefit to also include a sterol (preferably cholesterol) to theformulation as this allows a reduction in the total level of oil in theemulsion. This leads to a reduced cost of manufacture, improvement ofthe overall comfort of the vaccination, and also qualitative andquantitative improvements of the resultant immune responses, such asimproved IFN-γ production. Accordingly, the adjuvant system of thepresent invention typically comprises a ratio of metabolisableoil:saponin (w/w) in the range of 200:1 to 300:1, also the presentinvention can be used in a “low oil” form the preferred range of whichis 1:1 to 200:1, preferably 20:1 to 100:1, and most preferablysubstantially 48:1, this vaccine retains the beneficial adjuvantproperties of all of the components, with a much reduced reactogenicityprofile. Accordingly, the particularly preferred embodiments have aratio of squalene:QS21 (w/w) in the range of 1:1 to 250:1, also apreferred range is 20:1 to 200:1, preferably 20:1 to 100:1, and mostpreferably substantially 48:1. Preferably a sterol (most preferablycholesterol) is also included present at a ratio of saponin:sterol asdescribed herein.

The emulsion systems of the present invention preferably have a smalloil droplet size in the sub-micron range. Most preferably the oildroplet sizes will be in the range 120 to 750 nm, and most preferablyfrom 120-600 nm in diameter.

A particularly potent adjuvant formulation (for ultimate combinationwith AlPO4 in the immunogenic compositions of the invention) involves asaponin (preferably QS21), an LPS derivative (preferably 3D-MPL) and anoil emulsion (preferably squalene and alpha tocopherol in an oil inwater emulsion) as described in WO 95/17210 or in WO 99/12565 (inparticular adjuvant formulation 11 in Example 2, Table 1).

Examples of a TLR 2 agonist include peptidoglycan or lipoprotein.Imidazoquinolines, such as Imiquimod and Resiquimod are known TLR7agonists. Single stranded RNA is also a known TLR agonist (TLR8 inhumans and TLR7 in mice), whereas double stranded RNA and poly IC(polyinosinic-polycytidylic acid—a commercial synthetic mimetic of viralRNA). are exemplary of TLR 3 agonists. 3D-MPL is an example of a TLR4agonist whilst CPG is an example of a TLR9 agonist.

The immunogenic composition may comprise an antigen and animmunostimulant adsorbed onto a metal salt. Aluminium based vaccineformulations wherein the antigen and the immunostimulant 3-de-O-acylatedmonophosphoryl lipid A (3D-MPL), are adsorbed onto the same particle aredescribed in EP 0 576 478 B1, EP 0 689 454 B1, and EP 0 633 784 B1. Inthese cases then antigen is first adsorbed onto the aluminium saltfollowed by the adsorption of the immunostimulant 3D-MPL onto the samealuminium salt particles. Such processes first involve the suspension of3D-MPL by sonication in a water bath until the particles reach a size ofbetween 80 and 500 nm. The antigen is typically adsorbed onto aluminiumsalt for one hour at room temperature under agitation. The 3D-MPLsuspension is then added to the adsorbed antigen and the formulation isincubated at room temperature for 1 hour, and then kept at 4° C. untiluse.

In another process, the immunostimulant and the antigen are on separatemetal particles, as described in EP 1126876. The improved processcomprises the adsorption of immunostimulant, onto a metallic saltparticle, followed by the adsorption of the antigen onto anothermetallic salt particle, followed by the mixing of the discrete metallicparticles to form a vaccine. The adjuvant for use in the presentinvention may be an adjuvant composition comprising an immunostimulant,adsorbed onto a metallic salt particle, characterised in that themetallic salt particle is substantially free of other antigen.Furthermore, vaccines are provided by the present invention and arecharacterised in that the immunostimulant is adsorbed onto particles ofmetallic salt which are substantially free from other antigen, and inthat the particles of metallic salt which are adsorbed to the antigenare substantially free of other immunostimulant.

Accordingly, the present invention provides an adjuvant formulationcomprising immunostimulant which has been adsorbed onto a particle of ametallic salt, characterised in the composition is substantially free ofother antigen. Moreover, this adjuvant formulation can be anintermediate which, if such an adjuvant is used, is required for themanufacture of a vaccine. Accordingly there is provided a process forthe manufacture of a vaccine comprising admixing an adjuvant compositionwhich is one or more immunostimulants adsorbed onto a metal particlewith an antigen. Preferably, the antigen has been pre-adsorbed onto ametallic salt. Said metallic salt may be identical or similar to themetallic salt which is adsorbed onto the immunostimulant. Preferably themetal salt is an aluminium salt, for example Aluminium phosphate orAluminium hydroxide.

The present invention further provides for a vaccine compositioncomprising immunostimulant adsorbed onto a first particle of a metallicsalt, and antigen adsorbed onto a metallic salt, characterised in thatfirst and second particles of metallic salt are separate particles.

LPS or LOS derivatives or mutations or lipid A derivatives describedherein are designed to be less toxic (e.g. 3D-MPL) than nativelipopolysaccharides and are interchangeable equivalents with respect toany uses of these moieties described herein. They may be TLR4 ligands asdescribed above. Other such derivatives are described in WO020786737,WO9850399, WO0134617, WO0212258, WO03065806.

In one embodiment the adjuvant used for the compositions of theinvention comprises a liposome carrier (made by known techniques from aphospholipids (such as dioleoyl phosphatidyl choline [DOPC]) andoptionally a sterol [such as cholesterol]). Such liposome carriers maycarry lipid A derivatives [such as 3D-MPL—see above] and/or saponins(such as QS21—see above). In one embodiment the adjuvant comprises (per0.5 mL dose) 0.1-10 mg, 0.2-7, 0.3-5, 0.4-2, or 0.5-1 mg (e.g. 0.4-0.6,0.9-1.1, 0.5 or 1 mg) phospholipid (for instance DOPC), 0.025-2.5,0.05-1.5, 0.075-0.75, 0.1-0.3, or 0.125-0.25 mg (e.g. 0.2-0.3, 0.1-0.15,0.25 or 0.125 mg) sterol (for instance cholesterol), 5-60, 10-50, or20-30 μg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 μg) lipid A derivative(for instance 3D-MPL), and 5-60, 10-50, or 20-30 μg (e.g. 5-15, 40-50,10, 20, 30, 40 or 50 μg) saponin (for instance QS21).

This adjuvant is particularly suitable for elderly vaccine formulations.In one embodiment the vaccine composition comprising this adjuvantcomprises saccharide conjugates derived from at least all the followingserotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also compriseone or more from serotypes 3, 6A, 19A, and 22F), wherein the GMCantibody titre induced against one or more (or all) the vaccinecomponents 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferiorto that induced by the Prevnar® vaccine in human vaccinees.

In one embodiment the adjuvant used for the compositions of theinvention comprises an oil in water emulsion made from a metabolisableoil (such as squalene), an emulsifier (such as Tween 80) and optionallya tocol (such as alpha tocopherol). In one embodiment the adjuvantcomprises (per 0.5 mL dose) 0.5-15, 1-13, 2-11, 4-8, or 5-6 mg (e.g.2-3, 5-6, or 10-11 mg) metabolisable oil (such as squalene), 0.1-10,0.3-8, 0.6-6, 0.9-5, 1-4, or 2-3 mg (e.g. 0.9-1.1, 2-3 or 4-5 mg)emulsifier (such as Tween 80) and optionally 0.5-20, 1-15, 2-12, 4-10,5-7 mg (e.g. 11-13, 5-6, or 2-3 mg) tocol (such as alpha tocopherol).

This adjuvant may optionally further comprise 5-60, 10-50, or 20-30 μg(e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 μg) lipid A derivative (forinstance 3D-MPL).

These adjuvants are particularly suitable for infant or elderly vaccineformulations. In one embodiment the vaccine composition comprising thisadjuvant comprises saccharide conjugates derived from at least all thefollowing serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and mayalso comprise one or more from serotypes 3, 6A, 19A, and 22F), whereinthe GMC antibody titre induced against one or more (or all) the vaccinecomponents 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferiorto that induced by the Prevnar® vaccine in human vaccinees.

This adjuvant may optionally contain 0.025-2.5, 0.05-1.5, 0.075-0.75,0.1-0.3, or 0.125-0.25 mg (e.g. 0.2-0.3, 0.1-0.15, 0.25 or 0.125 mg)sterol (for instance cholesterol), 5-60, 10-50, or 20-30 μg (e.g. 5-15,40-50, 10, 20, 30, 40 or 50 μg) lipid A derivative (for instance3D-MPL), and 5-60, 10-50, or 20-30 μg (e.g. 5-15, 40-50, 10, 20, 30, 40or 50 g) saponin (for instance QS21).

This adjuvant is particularly suitable for elderly vaccine formulations.In one embodiment the vaccine composition comprising this adjuvantcomprises saccharide conjugates derived from at least all the followingserotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and may also compriseone or more from serotypes 3, 6A, 19A, and 22F), wherein the GMCantibody titre induced against one or more (or all) the vaccinecomponents 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferiorto that induced by the Prevnar® vaccine in human vaccinees.

In one embodiment the adjuvant used for the compositions of theinvention comprises aluminium phosphate and a lipid A derivative (suchas 3D-MPL). This adjuvant may comprise (per 0.5 mL dose) 100-750,200-500, or 300-400 μg Al as aluminium phosphate, and 5-60, 10-50, or20-30 μg (e.g. 5-15, 40-50, 10, 20, 30, 40 or 50 μg) lipid A derivative(for instance 3D-MPL).

This adjuvant is particularly suitable for elderly or infant vaccineformulations. In one embodiment the vaccine composition comprising thisadjuvant comprises saccharide conjugates derived from at least all thefollowing serotypes: 4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F (and mayalso comprise one or more from serotypes 3, 6A, 19A, and 22F), whereinthe GMC antibody titre induced against one or more (or all) the vaccinecomponents 4, 6B, 9V, 14, 18C, 19F and 23F is not significantly inferiorto that induced by the Prevnar® vaccine in human vaccinees.

The vaccine preparations containing immunogenic compositions of thepresent invention may be used to protect or treat a mammal susceptibleto infection, by means of administering said vaccine via systemic ormucosal route. These administrations may include injection via theintramuscular, intraperitoneal, intradermal or subcutaneous routes; orvia mucosal administration to the oral/alimentary, respiratory,genitourinary tracts. Intranasal administration of vaccines for thetreatment of pneumonia or otitis media is preferred (as nasopharyngealcarriage of pneumococci can be more effectively prevented, thusattenuating infection at its earliest stage). Although the vaccine ofthe invention may be administered as a single dose, components thereofmay also be co-administered together at the same time or at differenttimes (for instance pneumococcal saccharide conjugates could beadministered separately, at the same time or 1-2 weeks after theadministration of the any bacterial protein component of the vaccine foroptimal coordination of the immune responses with respect to eachother). For co-administration, the optional Th1 adjuvant may be presentin any or all of the different administrations. In addition to a singleroute of administration, 2 different routes of administration may beused. For example, saccharides or saccharide conjugates may beadministered IM (or ID) and bacterial proteins may be administered IN(or ID). In addition, the vaccines of the invention may be administeredIM for priming doses and IN for booster doses.

The content of protein antigens in the vaccine will typically be in therange 1-100 μg, preferably 5-50 μg, most typically in the range 5-25 μg.Following an initial vaccination, subjects may receive one or severalbooster immunizations adequately spaced.

Vaccine preparation is generally described in Vaccine Design (“Thesubunit and adjuvant approach” (eds Powell M. F. & Newman M. J.) (1995)Plenum Press New York). Encapsulation within liposomes is described byFullerton, U.S. Pat. No. 4,235,877.

The vaccines of the present invention may be stored in solution orlyophilized. Preferably the solution is lyophilized in the presence of asugar such as sucrose or lactose. It is still further preferable thatthey are lyophilized and extemporaneously reconstituted prior to use.Lyophilizing may result in a more stable composition (vaccine) and maypossibly lead to higher antibody titers in the presence of 3D-MPL and inthe absence of an aluminum based adjuvant.

In one aspect of the invention is provided a vaccine kit, comprising avial containing an immunogenic composition of the invention, optionallyin lyophilised form, and further comprising a vial containing anadjuvant as described herein. It is envisioned that in this aspect ofthe invention, the adjuvant will be used to reconstitute the lyophilisedimmunogenic composition.

Although the vaccines of the present invention may be administered byany route, administration of the described vaccines into the skin (ID)forms one embodiment of the present invention. Human skin comprises anouter “horny” cuticle, called the stratum corneum, which overlays theepidermis. Underneath this epidermis is a layer called the dermis, whichin turn overlays the subcutaneous tissue. Researchers have shown thatinjection of a vaccine into the skin, and in particular the dermis,stimulates an immune response, which may also be associated with anumber of additional advantages. Intradermal vaccination with thevaccines described herein forms a preferred feature of the presentinvention.

The conventional technique of intradermal injection, the “mantouxprocedure”, comprises steps of cleaning the skin, and then stretchingwith one hand, and with the bevel of a narrow gauge needle (26-31 gauge)facing upwards the needle is inserted at an angle of between 10-15°.Once the bevel of the needle is inserted, the barrel of the needle islowered and further advanced whilst providing a slight pressure toelevate it under the skin. The liquid is then injected very slowlythereby forming a bleb or bump on the skin surface, followed by slowwithdrawal of the needle.

More recently, devices that are specifically designed to administerliquid agents into or across the skin have been described, for examplethe devices described in WO 99/34850 and EP 1092444, also the jetinjection devices described for example in WO 01/13977; U.S. Pat. Nos.5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189,5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335,5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880,4,940,460, WO 97/37705 and WO 97/13537. Alternative methods ofintradermal administration of the vaccine preparations may includeconventional syringes and needles, or devices designed for ballisticdelivery of solid vaccines (WO 99/27961), or transdermal patches (WO97/48440; WO 98/28037); or applied to the surface of the skin(transdermal or transcutaneous delivery WO 98/20734; WO 98/28037).

When the vaccines of the present invention are to be administered to theskin, or more specifically into the dermis, the vaccine is in a lowliquid volume, particularly a volume of between about 0.05 ml and 0.2ml.

The content of antigens in the skin or intradermal vaccines of thepresent invention may be similar to conventional doses as found inintramuscular vaccines (see above). However, it is a feature of skin orintradermal vaccines that the formulations may be “low dose”.Accordingly the protein antigens in “low dose” vaccines are preferablypresent in as little as 0.1 to 10 μg, preferably 0.1 to 5 μg per dose;and the saccharide (preferably conjugated) antigens may be present inthe range of 0.01-1 μg, and preferably between 0.01 to 0.5 μg ofsaccharide per dose.

As used herein, the term “intradermal delivery” means delivery of thevaccine to the region of the dermis in the skin. However, the vaccinewill not necessarily be located exclusively in the dermis. The dermis isthe layer in the skin located between about 1.0 and about 2.0 mm fromthe surface in human skin, but there is a certain amount of variationbetween individuals and in different parts of the body. In general, itcan be expected to reach the dermis by going 1.5 mm below the surface ofthe skin. The dermis is located between the stratum corneum and theepidermis at the surface and the subcutaneous layer below. Depending onthe mode of delivery, the vaccine may ultimately be located solely orprimarily within the dermis, or it may ultimately be distributed withinthe epidermis and the dermis.

The present invention further provides an improved vaccine for theprevention or amelioration of Otitis media caused by Haemophilusinfluenzae by the addition of Haemophilus influenzae proteins, forexample protein D in free or conjugated form. In addition, the presentinvention further provides an improved vaccine for the prevention oramelioration of pneumococcal infection in infants (e.g., Otitis media),by relying on the addition of one or two pneumococcal proteins as freeor conjugated protein to the S. pneumoniae conjugate compositions of theinvention. Said pneumococcal free proteins may be the same or differentto any S. pneumoniae proteins used as carrier proteins. One or moreMoraxella catarrhalis protein antigens can also be included in thecombination vaccine in a free or conjugated form. Thus, the presentinvention is an improved method to elicit a (protective) immune responseagainst Otitis media in infants.

In another embodiment, the present invention is an improved method toelicit a (protective) immune response in infants (defined as 0-2 yearsold in the context of the present invention) by administering a safe andeffective amount of the vaccine of the invention [a paediatric vaccine].Further embodiments of the present invention include the provision ofthe antigenic S. pneumoniae conjugate compositions of the invention foruse in medicine and the use of the S. pneumoniae conjugates of theinvention in the manufacture of a medicament for the prevention (ortreatment) of pneumococcal disease.

In yet another embodiment, the present invention is an improved methodto elicit a (protective) immune response in the elderly population (inthe context of the present invention a patient is considered elderly ifthey are 50 years or over in age, typically over 55 years and moregenerally over 60 years) by administering a safe and effective amount ofthe vaccine of the invention, preferably in conjunction with one or twoS. pneumoniae proteins present as free or conjugated protein, which freeS. pneumoniae proteins may be the same or different as any S. pneumoniaeproteins used as carrier proteins.

A further aspect of the invention is a method of immunising a human hostagainst disease caused by S. pneumoniae and optionally Haemophilusinfluenzae infection comprising administering to the host animmunoprotective dose of the immunogenic composition or vaccine or kitof the invention. A further aspect of the invention is an immunogeniccomposition of the invention for use in

the treatment or prevention of disease caused by S. pneumoniae andoptionally Haemophilus influenzae infection.

A further aspect of the invention is use of the immunogenic compositionor vaccine or kit of the invention in the manufacture of a medicamentfor the treatment or prevention of diseases caused by S. pneumoniae andoptionally Haemophilus influenzae infection.

The terms “comprising”, “comprise” and “comprises” herein are intendedby the inventors to be optionally substitutable with the terms“consisting of”, “consist of” and “consists of”, respectively, in everyinstance.

Embodiments herein relating to “vaccine compositions” of the inventionare also applicable to embodiments relating to “immunogeniccompositions” of the invention, and vice versa.

All references or patent applications cited within this patentspecification are incorporated by reference herein.

In order that this invention may be better understood, the followingexamples are set forth.

These examples are for purposes of illustration only, and are not to beconstrued as limiting the scope of the invention in any manner.

EXAMPLES Example 1: Expression of Protein D

Haemophilus influenzae Protein D

Genetic Construction for Protein D Expression

Starting Materials

The Protein D Encoding DNA

Protein D is highly conserved among H. influenzae of all serotypes andnon-typeable strains. The vector pHIC348 containing the DNA sequenceencoding the entire protein D gene has been obtained from Dr. A.Forsgren, Department of Medical Microbiology, University of Lund, MalmoGeneral Hospital, Malmo, Sweden. The DNA sequence of protein D has beenpublished by Janson et al. (1991) Infect. Immun. 59: 119-125.

The Expression Vector pMG1

The expression vector pMG1 is a derivative of pBR322 (Gross et al.,1985) in which bacteriophage A derived control elements fortranscription and translation of foreign inserted genes were introduced(Shatzman et al., 1983). In addition, the Ampicillin resistance gene wasexchanged with the Kanamycin resistance gene.

The E. coli Strain AR58

The E. coli strain AR58 was generated by transduction of N99 with a P1phage stock previously grown on an SA500 derivative (galE::TN10,lambdaKil⁻ c1857 ΔH1). N99 and SA500 are E. coli K12 strains derivedfrom Dr. Martin Rosenberg's laboratory at the National Institute ofHealth.

The Expression Vector pMG 1

For the production of protein D, the DNA encoding the protein has beencloned into the expression vector pMG 1. This plasmid utilises signalsfrom lambdaphage DNA to drive the transcription and translation ofinserted foreign genes. The vector contains the promoter PL, operator OLand two utilisation sites (NutL and NutR) to relieve transcriptionalpolarity effects when N protein is provided (Gross et al., 1985).Vectors containing the PL promoter, are introduced into an E. colilysogenic host to stabilise the plasmid DNA. Lysogenic host strainscontain replication-defective lambdaphage DNA integrated into the genome(Shatzman et al., 1983). The chromosomal lambdaphage DNA directs thesynthesis of the cl repressor protein which binds to the OL repressor ofthe vector and prevents binding of RNA polymerase to the PL promoter andthereby transcription of the inserted gene. The cl gene of theexpression strain AR58 contains a temperature sensitive mutant so thatPL directed transcription can be regulated by temperature shift, i.e. anincrease in culture temperature inactivates the repressor and synthesisof the foreign protein is initiated. This expression system allowscontrolled synthesis of foreign proteins especially of those that may betoxic to the cell (Shimataka & Rosenberg, 1981).

The E. coli Strain AR58

The AR58 lysogenic E. coli strain used for the production of the proteinD carrier is a derivative of the standard NIH E. coli K12 strain N99 (F⁻su⁻ galK2, lacZ⁻ thr⁻). It contains a defective lysogenic lambdaphage(galE::TN10, lambdaKil⁻ c1857 ΔH1). The Kil⁻ phenotype prevents the shutoff of host macromolecular synthesis. The c1857 mutation confers atemperature sensitive lesion to the cl repressor. The ΔH1 deletionremoves the lambdaphage right operon and the hosts bio, uvr3, and chlAloci. The AR58 strain was generated by transduction of N99 with a P1phage stock previously grown on an SA500 derivative (galE::TN10,lambdaKil⁻ c1857 ΔH1). The introduction of the defective lysogen intoN99 was selected with tetracycline by virtue of the presence of a TN10transposon coding for tetracyclin resistance in the adjacent galE gene.

Construction of Vector pMGMDPPrD

The pMG 1 vector which contains the gene encoding the non-structural S1protein of Influenzae virus (pMGNSI) was used to construct pMGMDPPrD.The protein D gene was amplified by PCR from the pHIC348 vector (Jansonet al. 1991 Infect. Immun. 59:119-125) with PCR primers containing Ncoland Xbal restriction sites at the 5′ and 3′ ends, respectively. TheNcol/Xbal fragment was then introduced into pMGNS1 between Ncol and Xbalthus creating a fusion protein containing the N-terminal 81 amino acidsof the NS1 protein followed by the PD protein. This vector was labelledpMGNS1PrD.

Based on the construct described above the final construct for protein Dexpression was generated. A BamHI/BamHI fragment was removed frompMGNS1PrD. This DNA hydrolysis removes the NS1 coding region, except forthe first three N-terminal residues. Upon religation of the vector agene encoding a fusion protein with the following N-terminal amino acidsequence has been generated:

-----MDP SSHSSNMANT----- NS1                Protein D

The protein D does not contain a leader peptide or the N-terminalcysteine to which lipid chains are normally attached. The protein istherefore neither excreted into the periplasm nor lipidated and remainsin the cytoplasm in a soluble form.

The final construct pMG-MDPPrD was introduced into the AR58 host strainby heat shock at 37° C. Plasmid containing bacteria were selected in thepresence of Kanamycin. Presence of the protein D encoding DNA insert wasdemonstrated by digestion of isolated plasmid DNA with selectedendonucleases. The recombinant E. coli strain is referred to as ECD4.

Expression of protein D is under the control of the lambda P_(L)promoter/O_(L) Operator. The host strain AR58 contains atemperature-sensitive cl gene in the genome which blocks expression fromlambda P_(L) at low temperature by binding to O_(L). Once thetemperature is elevated cl is released from O_(L) and protein D isexpressed.

Small-Scale Preparation

At the end of the fermentation the cells are concentrated and frozen.

The extraction from harvested cells and the purification of protein Dwas performed as follows. The frozen cell culture pellet is thawed andresuspended in a cell disruption solution (Citrate buffer pH 6.0) to afinal OD₆₅₀=60. The suspension is passed twice through a high pressurehomogenizer at P=1000 bar. The cell culture homogenate is clarified bycentrifugation and cell debris is removed by filtration. In the firstpurification step the filtered lysate is applied to a cation exchangechromatography column (SP Sepharose Fast Flow). PD binds to the gelmatrix by ionic interaction and is eluted by a step increase of theionic strength of the elution buffer.

In a second purification step impurities are retained on an anionicexchange matrix (Q Sepharose Fast Flow). PD does not bind onto the geland can be collected in the flow through.

In both column chromatography steps fraction collection is monitored byOD. The flow through of the anionic exchange column chromatographycontaining the purified protein D is concentrated by ultrafiltration.

The protein D containing ultrafiltration retentate is finally passedthrough a 0.2 μm membrane.

Large Scale Preparation

The extraction from harvested cells and the purification of protein Dwas performed as follows. The harvested broth is cooled and directlypassed twice through a high pressure homogenizer at a Pressure of around800 bars.

In the first purification step the cell culture homogenate is dilutedand applied to a cation exchange chromatography column (SP Sepharose Bigbeads). PD binds to the gel matrix by ionic interaction and is eluted bya step increase of the ionic strength of the elution buffer andfiltrated.

In a second purification step impurities are retained on an anionicexchange matrix (Q Sepharose Fast Flow). PD does not bind onto the geland can be collected in the flow through.

In both column chromatography steps fraction collection is monitored byOD. The flow through of the anionic exchange column chromatographycontaining the purified protein D is concentrated and diafiltrated byultrafiltration.

The protein D containing ultrafiltration retentate is finally passedthrough a 0.2 μm membrane.

Example 1b: Expression of PhtD

The PhtD protein is a member of the pneumococcal histidine-triad (Pht)protein family characterized by the presence of histidine-triads (HXXHXHmotif). PhtD is a 838 aa-molecule and carries 5 histidine triads (seeMedImmune WO00/37105 SEQ ID NO: 4 for amino acid sequence and SEQ ID NO:5 for DNA sequence). PhtD also contains a proline-rich region in themiddle (amino acid position 348-380). PhtD has a 20 aa-N-terminal signalsequence with a LXXC motif.

Genetic Construct

The gene sequence of the mature MedImmune PhtD protein (from aa 21 to aa838) was transferred recombinantly to E. coli using the in-house pTCMP14vector carrying the pλ promoter. The E. coli host strain is AR58, whichcarries the c1857 thermosensitive repressor, allowing heat-induction ofthe promotor.

Polymerase chain reaction was realized to amplify the phtD gene from aMedImmune plasmid (carrying the phtD gene from Streptococcus pneumoniaestrain Norway 4 (serotype 4)—SEQ ID NO: 5 as described in WO 00/37105).Primers, specific for the phtD gene only, were used to amplify the phtDgene in two fragments. Primers carry either the NdeI and KpnI or theKpnI and XbaI restriction sites. These primers do not hybridize with anynucleotide from the vector but only with phtD specific gene sequences.An artificial ATG start codon was inserted using the first primercarrying the NdeI restriction site. The generated PCR products were theninserted into the pGEM-T cloning vector (Promega), and the DNA sequencewas confirmed. Subcloning of the fragments in the TCMP14 expressionvector was then realized using standard techniques and the vector wastransformed into AR58 E. coli.

PhtD Purification

PhtD purification is achieved as follows:

-   -   Growth of E. coli cells in the presence of Kanamycin: growth 30        hours at 30° C. then induction for 18 hours at 39.5° C.    -   Breakage of the E. coli cells from whole culture at OD±115 in        presence of EDTA 5 mM and PMSF 2 mM as protease inhibitors:        Rannie, 2 passages, 1000 bars.    -   Antigen capture and cells debris removal on expanded bed mode        Streamline Q XL chromatography at room temperature (20° C.); the        column is washed with NaCl 150 mM+Empigen 0.25% pH 6.5 and        eluted with NaCl 400 mM+Empigen 0.25% in 25 mM potassium        phosphate buffer pH 7.4.    -   Filtration on Sartobran 150 cartridge (0.45+0.2 μm)    -   Antigen binding on Zn⁺⁺ Chelating Sepharose FF IMAC        chromatography at pH 7.4 in presence of 5 mM imidazole at 4° C.;        the column is washed with Imidazole 5 mM and Empigen 1% and        eluted with 50 mM imidazole, both in 25 mM potassium phosphate        buffer pH 8.0.    -   Weak anion exchange chromatography in positive mode on Fractogel        EMD DEAE at pH 8.0 (25 mM potassium phosphate) at 4° C.; the        column is washed with 140 mM NaCl and eluted at 200 mM NaCl        while contaminants (proteins and DNA) remain adsorbed on the        exchanger.    -   Concentration and ultrafiltration with 2 mM Na/K phosphate pH        7.15 on 50 kDa membrane.    -   Sterilising filtration of the purified bulk on a Millipak-20 0.2        μm filter cartridge.

Example 1c: Expression of Pneumolysin

Pneumococcal pneumolysin was prepared and detoxified as described inWO2004/081515 and WO2006/032499.

Example 2

Preparation of Conjugates

It is well known in the art how to make purified pneumococcalpolysaccharides. For the purposes of these examples the polysaccharideswere made essentially as described in EP072513 or by closesly-relatedmethods. Before conjugation the polysaccharides may be sized bymicrofluidisation as described below.

The activation and coupling conditions are specific for eachpolysaccharide. These are given in Table 1. Sized polysaccharide (exceptfor PS5, 6B and 23F) was dissolved in NaCl 2M, NaCl 0.2M or in water forinjection (WFI). The optimal polysaccharide concentration was evaluatedfor all the serotypes. All serotypes except serotype 18C were conjugateddirectly to the carrier protein as detailed below. Two alternativeserotype 22F conjugates were made; one conjugated directly, one throughan ADH linker.

From a 100 mg/ml stock solution in acetonitrile or acetonitrile/water50%/50% solution, CDAP (CDAP/PS ratio 0.5-1.5 mg/mg PS) was added to thepolysaccharide solution. 1.5 minute later, 0.2M-0.3M NaOH was added toobtain the specific activation pH. The activation of the polysaccharidewas performed at this pH during 3 minutes at 25° C. Purified protein(protein D, PhtD, pneumolysin or DT) (the quantity depends on theinitial PS/carrier protein ratio) was added to the activatedpolysaccharide and the coupling reaction was performed at the specificpH for up to 2 hour (depending upon serotype) under pH regulation. Inorder to quench un-reacted cyanate ester groups, a 2M glycine solutionwas then added to the mixture. The pH was adjusted to the quenching pH(pH 9.0). The solution was stirred for 30 minutes at 25° C. and thenovernight at 2-8° C. with continuous slow stirring.

Preparation of 18C:

18C was linked to the carrier protein via a linker—Adipic aciddihydrazide (ADH) Polysaccharide serotype 18C was microfluidized beforeconjugation.

Derivatization of Tetanus Toxoid with EDAC

For derivatization of the tetanus toxoid, purified TT was diluted at 25mg/ml in 0.2M NaCl and the ADH spacer was added in order to reach afinal concentration of 0.2M. When the dissolution of the spacer wascomplete, the pH was adjusted to 6.2. EDAC(1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) was then added toreach a final concentration of 0.02M and the mixture was stirred for 1hour under pH regulation. The reaction of condensation was stopped byincreasing pH up to 9.0 for at least 30 minutes at 25° C. Derivatized TTwas then diafiltrated (10 kDa CO membrane) in order to remove residualADH and EDAC reagent.

TT_(AH) bulk was finally sterile filtered until coupling step and storedat −70° C.

Chemical Coupling of TT_(AH) to PS 18C

Details of the conjugation parameters can be found in Table 1.

2 grams of microfluidized PS were diluted at the defined concentrationin water and adjusted to 2M NaCl by NaCl powder addition.

CDAP solution (100 mg/ml freshly prepared in 50/50 v/v acetonitrile/WFI)was added to reach the appropriate CDAP/PS ratio.

The pH was raised up to the activation pH 9.0 by the addition of 0.3MNaOH and was stabilised at this pH until addition of TT_(AH).

After 3 minutes, derivatized TT_(AH) (20 mg/ml in 0.2 M NaCl) was addedto reach a ratio TT_(AH)/PS of 2; the pH was regulated to the couplingpH 9.0. The solution was left one hour under pH regulation.

For quenching, a 2M glycine solution, was added to the mixturePS/TT_(AH)/CDAP.

The pH was adjusted to the quenching pH (pH 9.0).

The solution was stirred for 30 min at 25° C., and then left overnightat 2-8° C. with continuous slow stirring.

PS22F_(AH)-PhtD Conjugate

In a second conjugation method for this saccharide (the first being thedirect PS22-PhtD conjugation method shown in Table 1), 22F was linked tothe carrier protein via a linker—Adipic acid dihydrazide (ADH).Polysaccharide serotype 22F was microfluidized before conjugation.

PS 22F Derivatization

Activation and coupling are performed at 25° C. under continuousstirring in a temperature-controlled waterbath.

Microfluidized PS22F was diluted to obtain a final PS concentration of 6mg/ml in 0.2M NaCl and the solution was adjusted at pH 6.05±0.2 with0.1N HCl.

CDAP solution (100 mg/ml freshly prepared in acetonitrile/WFI, 50/50)was added to reach the appropriate CDAP/PS ratio (1.5/1 ww).

The pH was raised up to the activation pH 9.00±0.05 by the addition of0.5M NaOH and was stabilised at this pH until addition of ADH.

After 3 minutes, ADH was added to reach the appropriate ADH/PS ratio(8.9/1 w/w); the pH was regulated to coupling pH 9.0. The solution wasleft for 1 hour under pH regulation.

The PS_(AH) derivative was concentrated and diafiltrated.

Coupling

PhtD at 10 mg/ml in 0.2M NaCl was added to the PS22F_(AH) derivative inorder to reach a PhtD/PS22F_(AH) ratio of 4/1 (w/w). The pH was adjustedto 5.0±0.05 with HCl. The EDAC solution (20 mg/ml in 0.1M Tris-HCl pH7.5) was added manually in 10 min (250 μl/min) to reach 1 mg EDAC/mgPS22F_(AH). The resulting solution was incubated for 150 min (though 60mins was also used) at 25° C. under stirring and pH regulation. Thesolution was neutralized by addition of 1M Tris-HCl pH 7.5 ( 1/10 of thefinal volume) and let 30 min at 25° C.

Prior to the elution on Sephacryl S400HR, the conjugate was clarifiedusing a 5 μm Minisart filter.

The resulting conjugate has a final PhtD/PS ratio of 4.1 (w/w), a freePS content below 1% and an antigenicity (α-PS/α-PS) of 36.3% andanti-PhtD antigenicity of 7.4%.

Purification of the Conjugates:

The conjugates were purified by gel filtration using a Sephacryl S400HRgel filtration column equilibrated with 0.15M NaCl (S500HR for 18C) toremove small molecules (including DMAP) and unconjugated PS and protein.Based on the different molecular sizes of the reaction components,PS-PD, PS-TT, PS-PhtD, PS-pneumolysin or PS-DT conjugates are elutedfirst, followed by free PS, then by free PD or free DT and finally DMAPand other salts (NaCl, glycine).

Fractions containing conjugates are detected by UV_(280 nm). Fractionsare pooled according to their Kd, sterile filtered (0.22 m) and storedat +2-8° C. The PS/Protein ratios in the conjugate preparations weredetermined.

Specific Activation/Coupling/Quenching Conditions of PS S.pneumoniae-Protein D/TT/DT/PhtD/Polyconjugates

Where “μfluid” appears in a row header, it indicates that the saccharidewas sized by microfluidisation before conjugation. Sizes of saccharidesfollowing microfluidisation are given in table 2.

TABLE 1 Specific activation/coupling/quenching conditions of PSS.pneumoniae-Protein D/TT/DT/PhtD/Plyconjugates 1 4 7F Serotype μfluidμfluid 5 6A 6B μfluid PS conc. 2.5 2.5 7.1 5.0 5.0 5.0 (mg/ml) PS WFIWFI WFI NaCl 2M NaCl 2M NaCl 2M dissolution PD conc. 10.0 10.0 5.0 5.05.0 10.0 (mg/ml) Initial PD/PS 1.5/1    1.5/1    1/1   1/1   1.1/1   1.2/1    Ratio (w/w) CDAP conc. 0.50 0.50 0.79 0.83 0.83 0.75 (mg/mg PS)pH_(a) = pH_(c) = pH_(q) 9.0/9.0/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.5/9.5/9.09.5/9.5/9.0 9.5/9.5/9.0 9V 14 18C 19A 19F 22F Serotype μfluid μfluidμfluid μfluid μfluid μfluid 23F PS conc. 5.0 5.0 4.5 15.0 9.0 6.0 2.38(mg/ml) PS NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 0.2M NaCl 2Mdissolution Carrier 10.0 10.0 20.0 10.0 20.0 10.0 5.0 protein conc. (TT)(Ply) (DT) (PhtD) (mg/ml) Initial carrier 1.2/1    1.2/1    2/1  2.5/1    1.5/1    3/1   1/1   protein/PS Ratio (w/w) CDAP conc. 0.500.75 0.75 1.5 1.5 1.5 0.79 (mg/mg PS) pH_(a) = pH_(c) = pH_(q)9.5/9.5/9.0 9.5/9.5/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.0 9.0/9.0/9.09.5/9.5/9.0 Note: pHa, c, q corresponds to the pH for activation,coupling and quenching, respectively

Characterisation:

Each conjugate was characterised and met the specifications described inTable 2. The polysaccharide content (μg/ml) was measured by theResorcinol test and the protein content (μg/ml) by the Lowry test. Thefinal PS/PD ratio (w/w) is determined by the ratio of theconcentrations.

Free Polysaccharide Content (%):

The free polysaccharide content of conjugates kept at 4° C. or stored 7days at 37° C. was determined on the supernatant obtained afterincubation with α-carrier protein antibodies and saturated ammoniumsulfate, followed by a centrifugation.

An α-PS/α-PS ELISA was used for the quantification of freepolysaccharide in the supernatant. The absence of conjugate was alsocontrolled by an α-carrier protein/α-PS ELISA.

Antigenicity:

The antigenicity on the same conjugates was analyzed in a sandwich-typeELISA wherein the capture and the detection of antibodies were α-PS andα-Protein respectively.

Free Protein Content (%):

Unconjugated carrier protein can be separated from the conjugate duringthe purification step. The content of free residual protein wasdetermined using size exclusion chromatography (TSK 5000-PWXL) followedby UV detection (214 nm). The elution conditions allowed separating thefree carrier protein and the conjugate. Free protein content inconjugate bulks was then determined versus a calibration curve (from 0to 50 g/ml of carrier protein). Free carrier protein in % was obtainedas follows: % free carrier=(free carrier (g/ml)/(Total concentration ofcorresponding carrier protein measured by Lowry (μg/ml)*100%).

Stability:

Molecular weight distribution (Kay) and stability was measured on aHPLC-SEC gel filtration (TSK 5000-PWXL) for conjugates kept at 4° C. andstored for 7 days at 37° C.

The 10/11/13/14-valent characterization is given in Table 2 (see commentthereunder).

The protein conjugates can be adsorbed onto aluminium phosphate andpooled to form the final vaccine.

Conclusion:

Immunogenic conjugates have been produced, that have since been shown tobe components of a promising vaccine.

TABLE 2 characteristics of the conjugates Carrier/ PS Conj. PS size PSFree PS Antigenicity Size Conjugates (Da × 10³) Ratio (Elisa) FreeCarrier (Elisa) (kDa) PS1-PD   349-382* 1.5-1.6 1.0%-1.2% 3.9%-4.8%87%-95% 1499-1715 PS4-PD  93-100* 1.5-1.6   4.7-6.5% 3.2%-4.0% 90%-96%1303-1606 PS5-PD***  367-443 0.80    8.7-11.2% 2.2%-3.8%  93%-108%1998-2352 PS6A-PD 1100-1540 0.61 4.5% Not done 45.9% Not done PS6B-PD***1069-1391 0.7-0.8  1.3-1.6% <2.0% 68%-75% 4778-5235 PS7F-PD   255-264*1.1-1.2 <1% <1.4%   58% 3907-4452 PS9V-PD   258-280* 1.3-1.5 <1% <1.3%67%-69% 9073-9572 PS14-PD   232-241* 1.4 <1% <1.5%   70% 3430-3779PS18C-TT   89-97* 2.2-2.4  1.5-2.2%   <4% 46%-56% 5464-6133 PS19A-Ply*151 3.2 <1%   29% PS19F-DT  133-143* 1.4-1.5 4.1%-5.9% <1.2%-<1.3%82%-88% 2059-2335 PS22F-PhtD* 159-167 2.17 5.8 Not done   37% Not donePS22F-AHPhtD* 159-167 3.66-4.34 <1% Not done   28-31% Not donePS23F-PD***  914-980 0.5  1.4-1.9% 3.7%-4.9% 137%-154% 2933-3152 *PSsize following microfluidization of the native PS

A 10 valent vaccine was made by mixing serotype 1, 4, 5, 6B, 7F, 9V, 14,18C, 19F and 23F conjugates (e.g. at a dose of 1, 3, 1, 1, 1, 1, 1, 3,3, 1 μg of saccharide, respectively per human dose). An 11 valentvaccine was made by further adding the serotype 3 conjugate from Table 5(e.g. at 1 μg of saccharide per human dose). A 13 valent vaccine wasmade by further adding the serotypes 19A and 22F conjugates above (with22F either directly linked to PhtD, or alternatively through an ADHlinker) [e.g. at a dose of 3 g each of saccharide per human dose]. A 14valent vaccine may be made by further adding the serotype 6A conjugateabove [e.g. at a dose of 1 μg of saccharide per human dose.

Example 3: Evidence that Inclusion of Haemophilus Influenzae Protein Din an Immunogenic Composition of the Invention can Provide ImprovedProtection Against Acute Otitis Media (AOM)

Study Design.

The study used an 11Pn-PD vaccine—comprising serotypes 1, 3, 4, 5, 6B,7F, 9V, 14, 18C, 19F and 23F each conjugated to protein D from H.influenzae (refer to Table 5 in Example 4). Subjects were randomizedinto two groups to receive four doses of either the 11 Pn-PD vaccine orHavrix at approximately 3, 4, 5 and 12-15 months of age. All subjectsreceived GSK Biologicals' Infanrix-hexa (DTPa—HBV-IPV/Hib) vaccineconcomitantly at 3, 4 and 5 months of age. Infanrix-hexa is acombination of Pediarix and Hib mixed before administration. Efficacyfollow-up for the “According-to-Protocol” analysis started 2 weeks afteradministration of the third vaccine dose and continued until 24-27months of age. Nasopharyngeal carriage of S. pneumoniae and H.influenzae was evaluated in a selected subset of subjects.

Parents were advised to consult the investigator if their child wassick, had ear pain, spontaneous perforation of the tympanic membrane orspontaneous ear discharge. If the investigator suspected an episode ofAOM, the child was immediately referred to an Ear, Nose and Throat (ENT)specialist for confirmation of the diagnosis.

A clinical diagnosis of AOM was based on either the visual appearance ofthe tympanic membrane (i.e. redness, bulging, loss of light reflex) orthe presence of middle ear fluid effusion (as demonstrated by simple orpneumatic otoscopy or by microscopy). In addition, at least two of thefollowing signs or symptoms had to be present: ear pain, ear discharge,hearing loss, fever, lethargy, irritability, anorexia, vomiting, ordiarrhea. If the ENT specialist confirmed the clinical diagnosis, aspecimen of middle ear fluid was collected by tympanocentesis forbacteriological testing.

For subjects with repeated sick visits, a new AOM episode was consideredto have started if more than 30 days had elapsed since the beginning ofthe previous episode. In addition, an AOM episode was considered to be anew bacterial episode if the isolated bacterium/serotype differed fromthe previous isolate whatever the interval between the two consecutiveepisodes.

Trial Results

A total of 4968 infants were enrolled, 2489 in the 11 Pn-PD group and2479 in the control group. There were no major differences in thedemographic characteristics or risk factors between the two groups.

Clinical Episodes and AOM Case Definition

During the per protocol follow-up period, a total of 333 episodes ofclinical AOM were recorded in the 11Pn-PD group and 499 in the controlgroup.

Table 3 presents the protective efficacy of the 11Pn-PD vaccine and both7-valent vaccines previously tested in Finland (Eskola et al N Engl JMed 2001; 344: 403-409 and Kilpi et al Clin Infect Dis 2003 37:1155-64)against any episode of AOM and AOM caused by different pneumococcalserotypes, H. influenzae, NTHi and M. catarrhalis. Statisticallysignificant and clinically relevant reduction by 33.6% of the overallAOM disease burden was achieved with 11Pn-PD, irrespective of theetiology (table 3).

The overall efficacy against AOM episodes due to any of the 11pneumococcal serotypes contained in the 11Pn-PD vaccine was 57.6% (table3).

Another important finding in the current study is the 35.6% protectionprovided by the 11Pn-PD vaccine against AOM caused by H. influenzae (andspecifically 35.3% protection provided by NTHi). This finding is ofmajor clinical significance, given the increased importance of H.influenzae as a major cause of AOM in the pneumococcal conjugate vaccineera. In line with the protection provided against AOM, the 11Pn-PDvaccine also reduced nasopharyngeal carriage of H. influenzae followingthe booster dose in the second year of life. These findings are incontrast with previous observations in Finland where, for both 7-valentpneumococcal conjugate vaccines, an increase in AOM episodes due to H.influenzae was observed, (Eskola et al and Kilpi et al) as evidence ofetiological replacement.

A clear correlation between protection against AOM episodes due to Hiand antibody levels against the carrier Protein D could not beestablished, as post-primary anti-PD IgG antibody concentrations in11Pn-PD vaccinees, that remained Hi AOM episode-free, were essentiallythe same as post-primary anti-PD IgG antibody levels measured in 11Pn-PDvaccinees that developed at least one Hi AOM episode during the efficacyfollow-up period. However, although no correlation could be establishedbetween the biological impact of the vaccine and the post-primary IgGanti-PD immunogenicity, it is reasonable to assume that the PD carrierprotein, which is highly conserved among H. influenzae strains, hascontributed to a large extent in the induction of the protection againstHi.

The effect on AOM disease was accompanied by an effect on nasopharyngealcarriage that was of similar magnitude for vaccine serotype pneumococciand H. influenzae (FIG. 1). This reduction of the nasopharyngealcarriage of H. influenzae in the PD-conjugate vaccinees supports thehypothesis of a direct protective effect of the PD-conjugate vaccineagainst H. influenzae, even if the protective efficacy could not becorrelated to the anti-PD IgG immune responses as measured by ELISA.

In a following experiment a chinchilla otitis media model was used withserum pools from infants immunised with the 11 valent formulation ofthis example or with the 10 valent vaccine of Example 2 (see also Table1 and 2 and comments thereunder). Both pools induce a significantreduction of the percentage of animals with otitis media versus thepre-immune serum pool. There is no significant difference between the 10and 11 valent immune pools. This demonstrates that both vaccines have asimilar potential to induce protection against otitis media caused bynon typeable H. influenzae in this model.

TABLE 3 11Pn-PD Prevnar in FinOM^((Eskola et al)) 7v-OMP inFinOM^((Kilip et al)) n VE n VE n VE 11Pn- 95% Cl 7v- 95% Cl 7v- 95% ClType of AOM episode PD Control % LL UL CRM Control % LL UL OMP Control %LL UL N 2455 2452 786 794 805 794 Any AOM 333 499 33.6 20.8 44.3 12511345 6 −4 16 1364 1345 −1 −12 10 Any AOM with MEF 322 474 32.4 19.0 43.61177 1267 7 −5 17 1279 1267 0 −12 10 Culture confirmed 92 189 51.5 36.862.9 271 414 34 21 45 314 414 25 11 37 pneumococcus Vaccine pneumococcal60 141 57.6 41.4 69.3 107 250 57 44 67 110 250 56 44 66 serotypes^((*))Other bacterial pathogens H. influenzae 44 68 35.6 3.8 57.0 315 287 −11−34 8 315 287 −9 −32 10 Non-typeable H. influenzae 41 63 35.3 1.8 57.4NP NP NP NP NP NP NP NC NP NP (NTH) M. catarrhalis 31 34 9.4 −52.5 46.1379 381 −1 −19 15 444 381 −16 −36 2 NP: Not published; N: number ofsubjects in ATP efficacy cohort; n: number of episodes *Vaccinepneumococcal serotypes: for 11Pn-PD = 11 serotypes, for Prevnar and7v-OMP = 7 serotypes MEF = Middle ear fluid

Example 4

Selection of Carrier Protein for Serotype 19F

ELISA Assay Used

The 22F inhibition ELISA method was essentially based on an assayproposed in 2001 by Concepcion and Frasch and was reported by Henckaertset al., 2006, Clinical and Vaccine Immunology 13:356-360. Briefly,purified pneumococcal polysaccharides were mixed with methylated humanserum albumin and adsorbed onto Nunc Maxisorp™ (Roskilde, DK) highbinding microtitre plates overnight at 4° C. The plates were blockedwith 10% fetal bovine serum (FBS) in PBS for 1 hour at room temperaturewith agitation. Serum samples were diluted in PBS containing 10% FBS, 10μg/mL cell-wall polysaccharide (SSI) and 2 μg/mL of pneumococcalpolysaccharide of serotype 22F (ATCC), and further diluted on themicrotitre plates with the same buffer. An internal reference calibratedagainst the standard serum 89-SF using the serotype-specific IgGconcentrations in 89-SF was treated in the same way and included onevery plate. After washing, the bound antibodies were detected usingperoxidase-conjugated anti-human IgG monoclonal antibody (StratechScientific Ltd., Soham, UK) diluted in 10% FBS (in PBS), and incubatedfor 1 hour at room temperature with agitation. The color was developedusing ready-to-use single component tetramethylbenzidine peroxidaseenzyme immunoassay substrate kit (BioRad, Hercules, Calif., US) in thedark at room temperature. The reaction was stopped with H2SO4 0.18 M,and the optical density was read at 450 nm. Serotype-specific IgGconcentrations (in μg/mL) in the samples were calculated by referencingoptical density points within defined limits to the internal referenceserum curve, which was modelized by a 4-parameter logistic log equationcalculated with SoftMax Pro™ (Molecular Devices, Sunnyvale, Calif.)software. The cut-off for the ELISA was 0.05 μg/mL IgG for all serotypestaking into account the limit of detection and the limit ofquantification.

Opsonophagocytosis Assay

At the WHO consultation meeting in June 2003, it was recommended to usean OPA assay as set out in Romero-Steiner et al Clin Diagn Lab Immunol2003 10 (6): pp 1019-1024. This protocol was used to test the OPAactivity of the serotypes in the following tests.

Preparation of Conjugates

In studies 11Pn-PD&Di-001 and 11Pn-PD&Di-007, three 11-valent vaccineformulations (Table 4) were included in which 3 μg of the 19Fpolysaccharide was conjugated to diphtheria toxoid (19F-DT) instead of 1μg polysaccharide conjugated to protein D (19F-PD). Conjugationparameters for the studies 11Pn-PD, 11 Pn-PD&Di-001 and 11 Pn-PD&Di-007are disclosed in Tables 5, 6 and 7 respectively.

Anti-pneumococcal antibody responses and OPA activity against serotype19F one month following primary vaccination with these 19F-DTformulations are shown in Table 8 and 9 respectively.

Table 10 shows 22F-ELISA antibody concentrations and percentages ofsubjects reaching the 0.2 μg/mL threshold before and after 23-valentplain polysaccharide booster vaccination.

The opsonophagocytic activity was shown to be clearly improved forantibodies induced with these 19F-DT formulations as demonstrated byhigher seropositivity rates (opsonophagocytic titers >1:8) and OPA GMTsone month following primary vaccination (Table 9). One month after23-valent plain polysaccharide booster vaccination, opsonophagocyticactivity of 19F antibodies remained significantly better for childrenprimed with 19F-DT formulations (Table 11).

Table 12 presents immunogenicity data following a 11Pn-PD booster dosein toddlers previously primed with 19F-DT or 19F-PD conjugates comparedto a 4^(th) consecutive dose of Prevnar®. Given the breakthrough casesreported after the introduction of Prevnar® in the US, the improvedopsonophagocytic activity against serotype 19F when conjugated to the DTcarrier protein may be an advantage for the candidate vaccine.

Table 13 provides ELISA and OPA data for the 19F-DT conjugate withrespect to the cross-reactive serotype 19A. It was found that 19F-DTinduces low but significant OPA activity against 19A.

TABLE 4 Pneumococcal conjugate vaccine formulations used in clinicalstudies. Pneumococcal serotype μg/carrier protein Al³⁺ Formulation 1 3 45 6B 7F 9V 14 18C 19F 23F mg 11Pn-PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD 1/PD1/PD 1/PD 1/PD 1/PD <0.8 19F-DT Form 1 3/PD 3/PD 3/PD 3/PD 10/DT 3/PD3/PD 3/PD 3/PD 3/DT 5/DT ≤0.35 19F-DT Form 2 3/PD 2/PD 2/PD 3/PD 5/DT3/PD 2/PD 2/PD 2/PD 3/DT 5/DT ≤0.35 19F-DT Form 3 3/PD 3/PD 3/PD 3/PD3/PD 3/PD 3/PD 3/PD 3/PD 3/DT 3/PD =0.5

TABLE 5 Specific activation/coupling/quenching conditions of PS S.pneumoniae-Protein D/TT/DT conjugates 1 3 4 5 6B 7F Serotype Nativeμfluid Native Native Native Native PS 1.5 2 2.0 7.5 5.5 3.0 conc.(mg/ml)PS dissolution NaCl NaCl WFI WFI NaCl NaCl 150 mM 2M 2M 2M PD 5.0 5.05.0 5.0 5.0 5.0 conc.(mg/ml) Initial PS/PD 1/0.7 1/1 1/1 1/1 1/1 1/1Ratio (w/w) CDAP conc. 0.75 0.75 0.75 0.75 0.75 0.75 (mg/mg PS) pH_(a) =pH_(c) = pH_(q) 9.0/9.0/ 9.0 9.5/9.5/ 9.0 8.8/8.8/ 9.0 9.0/9.0/ 9.09.5/9.5/ 9.0 9.0/9.0/ 9.0 Coupling time 60 mins 60 mins 45 mins 40 mins60 mins 60 mins 9V 14 18C 19F 23F Serotype Native Native Native NativeNative PS 1.75 2.5 1.75 4.0 2.5 conc.(mg/ml) PS dissolution NaCl 2M NaCl2M WFI NaCl 2M NaCl 2M PD 5.0 5.0 5.0 5.0 5.0 conc.(mg/ml) Initial PS/PD1/0.75 1/0.75 1/1.2 1/1 1/1 Ratio (w/w) CDAP conc. 0.75 0.75 0.75 0.750.75 (mg/mg PS) pH_(a) = pH_(c) = pH_(q) 8.5/8.5/ 9.0 9.0/9.0/ 9.09.0/9.0/ 9.0 9.5/9.5/ 9.0 9.5/9.5/ 9.0 Coupling time 60 mins 60 mins 45mins 30 mins 60 mins

TABLE 6 Specific activation/coupling/quenching conditions of PS S.pneumoniae-Protein D/DTconjugates for the 11 Pn-PD&Di-001 study 1 3 4 56B 7F Serotype μfluid μfluid μfluid μfluid μfluid Native PS 4 2.0 2.57.5 10 3.0 conc.(mg/ml) PS dissolution NaCl 2M NaCl 2M NaCl 2M NaCl 2MNaCl 2M NaCl 2M PD 10.0 5.0 5.0 5.0 20 (DT) 5.0 conc.(mg/ml) NaCl 2MNaCl 2M Initial PD/PS 1.2/1 1/1 1/1 1/1 1.5/1 1/1 Ratio (w/w) CDAP conc.1.50 0.75 1.5 2 1.5 0.75 (mg/mg PS) pH_(a) = pH_(c) = pH_(q) 9.0/9.0/9.0 9.5/9.5/ 9.0 9.5/9.5/ 9.0 9.0/9.0/ 9.0 9.5/9.5/ 9.0 9/9/9 Couplingtime 60 mins 60 mins 60 mins 60 mins 60 mins 60 mins 9V 14 18C 19F 23FSerotype Native Native μfluid μfluid μfluid PS 1.75 2.5 5.0 9.0 10conc.(mg/ml) PS dissolution NaCl 2M NaCl 2M NaCl 2M NaCl 2M NaCl 2MCarrier protein 5.0 5.0 5.0 20 (DT) 10 (DT) conc.(mg/ml) Initial carrier0.75/1 0.75/1 1.2/1 1.5/1 1.5/1 protein/PS Ratio (w/w) CDAP conc. 0.750.75 1.5 1.5 0.75 (mg/mg PS) pH_(a) = pH_(c) = pH_(q) 8.5/8.5/ 9.09.0/9.0/ 9.0 9.0/9.0/ 9.0 9.0/9.0/ 9.0 9.5/9.5/9.0 Coupling time 60 mins60 mins 30 mins 60 mins 60 mins

TABLE 7 Specific activation/coupling/quenching conditions of PS S.pneumoniae-Protein D/DTconjugates for the 11 Pn-PD&Di-007 study 1 3 4 56B 7F Serotype Native μfluid Native Native Native μfluid PS 1.5 2.0 27.5 5.5 5.0 conc.(mg/ml) PS NaCl NaCl WFI WFI NaCl NaCl dissolution 150mM 2M 2M 2M PD 5.0 5.0 5.0 5.0 5 10 conc.(mg/ml) Initial PD/PS 0.7/1 1/11 1/1 1/1 1.2/1 Ratio (w/w) CDAP conc. 0.75 0.75 0.75 0.75 0.75 0.75(mg/mg PS) pH_(a) = pH_(c) = pH_(q) 9.0/9.0/ 9.0 9.5/9.5/ 9.0 8.8/8.8/9.0 9.0/9.0/ 9.0 9.5/9.5/ 9.0 9.5./ 9.5/9 Coupling time 60 mins 60 mins45 mins 40 mins 60 mins 60 mins 9V 14 18C 19F 19F 23F Serotype μfluidμfluid Native μfluid μfluid μfluid PS 5.0 5.0 1.75 9.0 10.0 9.5conc.(mg/ml) PS dissolution NaCl 2M NaCl 2M WFI NaCl 2M NaCl 2M NaCl 2MCarrier protein 10 10.0 5.0 20 (DT) 5.0 (PD) 10 conc.(mg/ml) Initialcarrier 1.2/1 1.2/1 1.2/1 1.5/1 1.2/1 1/1 protein/PS Ratio (w/w) CDAPconc. 0.5 0.75 0.75 1.5 0.75 0.75 (mg/mg PS) pH_(a) = pH_(c) = pH_(q)9.5/9.5/ 9.0 9.5/9.5/ 9.0 9.0/9.0/ 9.0 9.0/9.0/ 9.0 9.0/9.0/ 9.0 9.5/9.5/9.0 Coupling time 60 mins 60 mins 45 mins 120 mins 120 mins 60 mins

TABLE 8 Percentage of subjects with 19F antibody concentration ≥0.20μg/mL and 19F antibody Geometric mean antibody concentrations (GMCs with95% Cl; μg/mL) one month following 1 μg 19F-PD, 3 μg 19F-DT or Prevnar(2 μg 19F-CRM) primary vaccination (Total cohort) 11Pn-PD&Di-001(22F-ELISA) 11Pn-PD&Di-007 (22F-ELISA) % ≥.20 μg/mL GMC (μg/mL) % ≥0.20μg/mL GMC (μg/mL) Group N (95% Cl) (95% Cl) N (95% Cl) (95% Cl) 11Pn-PD152 98.7 1.93 50 100 2.78 (95.3-99.8) (1.67-2.22) (92.9-100) (2.31-3.36)19F-DT Form 1^(Γ) 146 99.3 2.88 — — — (96.2-100) (2.45-3.38) 19F-DT Form2^(Γ) 150 96.0 2.43 — — — (91.5-98.5) (2.01-2.94) 19F-DT Form 3^(Γ) — —— 50 96.0 3.70 (86.3-99.5) (2.58-5.30) Prevnar 148 98.6 2.98 41 97.62.91 (95.2-99.8) (2.60-3.41) (87.1-99.9) (2.15-3.94) ^(Γ)The compositionof the different formulations is provided in table 4.

TABLE 9 Percentage of subjects with 19F OPA titer ≥1:8 and 19F OPA GMTsone month following primary vaccination with 1 μg 19F-PD, 3 μg 19F- DTor Prevnar (2 μg 19F-CRM) (Total cohort) 11Pn-PD&Di-001 11Pn-PD&Di-007≥1:8 GMT ≥1:8 GMT Group N (95% Cl) (95% Cl) N (95% Cl) (95% Cl) 11Pn-PD136 84.6 77.8 46 95.7 167.8 (77.4-90.2) (58.1-104.4) (85.2-99.5)(118.1-238.6) 19F-DT 137 95.6 263.2 — — Form 1^(Γ) (90.7-98.4)(209.4-330.7) 19F-DT 139 92.1 218.9 — — — Form 2^(Γ) (86.3-96.0)(166.5-287.9) 19F-DT — — — 49 91.8 403.1 Form 3^(Γ) (80.4-97.7)(225.7-719.9) Prevnar 131 86.3 82.6 38 81.6 65.0 (79.2-91.6)(61.1-111.6) (65.7-92.3) (37.7-112.2) ^(Γ)The composition of thedifferent formulations is provided in Table4.

TABLE 10 Percentage of subjects with 19F antibody concentration ≥0.20μg/mL and 19F antibody GMCs (μg/mL) prior to and one month following 23-valent plain polysaccharide booster in children primed with 1 μg 19F-PD, 3 μg 19F-DT or Prevnar (2 μg 19F-CRM) (Total cohort) 11Pn-PD&Di-002(22F ELISA) Prior to booster vaccination One month post 23-valent PSbooster % ≥0.20 μg/mL GMC (μg/ml) % ≥0.20 μg/mL GMC (μg/ml) Primarygroup N (95% Cl) (95% Cl) N (95% Cl) (95% Cl) 11Pn-PD 70 77.1 0.67 6794.0 11.50 (65.6-86.3) (0.45-0.98) (85.4-98.3) (7.76-17.03) 19F-DT Form1^(Γ) 68 91.2 0.71 69 98.6 14.50 (81.8-96.7) (0.54-0.94) (92.2-100)(10.47-20.07) 19F-DT Form 2^(Γ) 74 81.1 0.59 72 95.8 9.90 (70.3-89.3)(0.43-0.80) (88.3-99.1) (6.74-14.54) Prevnar 65 64.6 0.40 67 100 9.40(51.8-76.1) (0.27-0.60) (94.6-100) (6.95-12.71) ^(Γ)The composition ofthe different formulations is provided in Table4.

TABLE 11 Percentage of subjects with 19F OPA titer ≥1:8 and 19F OPA GMTsprior to and one month following 23-valent plain polysaccharide boosterin children primed with 1 μg 19F-PD, 3 μg 19F-DT or Prevnar (2 μg19F-CRM) (Total cohort) 11Pn-PD&Di-002 Prior to booster vaccination Onemonth post 23-valent PS booster Primary % ≥1:8 GMT % ≥1:8 GMT group N(95% Cl) (95% Cl) N (95% Cl) (95% Cl) 11Pn-PD 29 27.6 10.9 28 82.1 408.0(12.7-47.2) (5.0-23.7) (63.1-93.9) (157.3-1058.3) 19F-DT 19 47.4 18.1 1894.4 1063.8 Form 1^(Γ) (24.4-71.1) (7.2-45.7) (72.7-99.9) (386.6-2927.5)19F-DT 27 33.3 8.5 28 100 957.6 Form 2^(Γ) (16.5-54.0) (4.7-15.3)(87.7-100) (552.8-1659.0) Prevnar 24 12.5 8.1 23 82.6 380.9 (2.7-32.4)(3.4-19.6) (61.2-95.0) (133.2-1089.5) ^(Γ)The composition of thedifferent formulations is provided in Table4.

TABLE 12 Percentage of subjects with antibody concentrations ≥0.2 μg/mL,OPA ≥1:8 and GMCs/GMTs against 19F pneumococci one month following11Pn-PD or Prevnar booster in children primed with 1 μg 19F-PD, 3 μg19F-DT or Prevnar (2 μg 19F-CRM) (Total cohort) 11Pn-PD&Di-002 22F-ELISAassay OPA assay Primary % ≥0.20 μg/mL GMC (μg/ml) % ≥1:8 (95% GMT (95%group N (95% Cl) (95% Cl) N Cl) Cl) 11Pn-PD 70 100 4.52 21 100 255.6(94.9-100) (3.7-5.5) (83.9-100) (135.5-481.9) 19F-DT 66 98.5 3.45 2395.7 374.0 Form 1^(Γ) (91.8-100) (2.8-4.3) (78.1-99.9) (192.6-726.2)19F-DT 70 98.6 3.80 29 96.6 249.1 Form 2^(Γ) (92.3-100) (2.9-4.9)(82.2-99.9) (144.7-428.7) Prevnar 69 97.1 2.56 31 96.8 528.7 (89.9-99.6)(2.0-3.3) (83.3-99.9) (319.4-875.2) ^(Γ)The composition of the differentformulations is provided in Table4.

TABLE 13 Percentage of subjects with antibody concentrations ≥0.2 μg/mL,OPA ≥1:8 and GMCs/GMTs against 19A pneumococci one month followingprimary vaccination with 1 μg 19F-PD, 3 μg 19F-DT or Prevnar (2 μg19F-CRM) (Total cohort) 11Pn-PD&Di-001 22F-ELISA assay OPA assay % ≥0.20μg/mL GMC (μg/mL) % ≥1:8 GMT Group N (95% Cl) (95% Cl) N (95% Cl) (95%Cl) 11Pn- 45 28.9 0.09 52 7.7 5.2 PD (16.4-44.3) (0.07-0.11) (2.1-18.5)(4.0-6.8) 19F-DT 51 29.4 0.11 59 27.1 12.4 Form 2^(Γ) (17.5-43.8)(0.08-0.16) (16.4-40.3) (7.6-20.3) Prevnar 55 18.2 0.10 61 3.3 4.6(9.1-30.9) (0.08-0.12) (0.4-11.3) (3.8-5.6) ^(Γ)The composition of thedifferent formulations is provided in Table4

Example 5: Adjuvant Experiments in Preclinical Models: Impact on theImmunogenicity of Pneumococcal 11-Valent Polysaccharide Conjugates inElderly Rhesus Monkeys

To optimize the response elicited to conjugate pneumococcal vaccines inthe elderly population, GSK formulated an 11-valent polysaccharide (PS)conjugate vaccine with a novel adjuvant Adjuvant C—see below.

Groups of 5 elderly Rhesus monkeys (14 to 28 years-old) were immunizedintramuscularly (IM) at days 0 and 28 with 500 μl of either 11-valent PSconjugates adsorbed onto 315 μg of AlPO4 or 11-valent PS conjugatesadmixed with Adjuvant C.

In both vaccine formulations, the 11-valent PS conjugates were eachcomposed of the following conjugates PS1-PD, PS3-PD, PS4-PD, PS5-PD,PS7F-PD, PS9V-PD, PS14-PD, PS18C-PD, PS19F-PD, PS23F-DT and PS6B-DT. Thevaccine used was ⅕ dose of of the human dose of the vaccine (5 μg ofeach saccharide per human dose except for 6B [10 μg]) conjugatedaccording to Table 6 conditions (Example 4), except 19F was madeaccording to the following CDAP process conditions: sized saccharide at9 mg/ml, PD at 5 mg/ml, an initial PD/PS ratio of 1.2/1, a CDAPconcentration of 0.75 mg/mg PS, pHa=pHc=pHq 9.0/9.0/9.0 and a couplingtime of 60 min.

Anti-PS ELISA IgG levels and opsono-phagocytosis titres were dosed insera collected at day 42. Anti-PS3 memory B cell frequencies weremeasured by Elispot from peripheral blood cells collected at day 42.

According to the results shown here below, Adjuvant C significantlyimproved the immunogenicity of 11-valent PS conjugates versus conjugateswith AlPO4 in elderly monkeys. The novel adjuvant enhanced the IgGresponses to PS (FIG. 1) and the opsono-phagocytosis antibody titres(Table 14). There was also supportive evidence that the frequency ofPS3-specific memory B cells is increased by the use of Adjuvant C (FIG.2).

TABLE 14 Conjugate immunogenicity in elderly Rhesus monkeys (post-IIopsono- phagocytosis titres) PS1 PS3 PS4 PS5 PS6B PS7F PS9V PS14 PS18CPS19F PS23F 11-valent Pre-immune <8 5 <8 5 <8 16 <8 <8 <8 <8 <8 AlPO4day 14 post II 8 181 64 49 64 4096 42 37 169 64 <64 11 valent Pre-immune5 9 <8 5 8 37 <8 <8 <8 <8 <8 Adj-C day 14 post II 776 1351 891 676 620816384 111 161 7132 2048 <64

B Cell Elispot

The principle of the assay relies on the fact that memory B cells matureinto plasma cells in vitro following cultivation with CpG for 5 days. Invitro generated antigen-specific plasma cells can be easily detected andtherefore be enumerated using the B-cell elispot assay. The number ofspecific plasma cells mirrors the frequency of memory B cells at theonset of the culture.

Briefly, in vitro generated plasma cells are incubated in culture platescoated with antigen. Antigen-specific plasma cells form antibody/antigenspots, which are detected by conventional immuno-enzymatic procedure andenumerated as memory B cells. In the present study, Polysaccharides havebeen used to coat culture plates in order to enumerate respective memoryB cells. Results are expressed as a frequency of PS specific memory Bcells within a million of memory B cells.

The study shows that Adjuvant C may be able to alleviate the knownproblem of PS3 boostability (see 5th International Symposium onPneumococci and Pneumococcal Diseases, April 2-6 2006, Alice Springs,Central Australia.

Specificities of immune responses against a serotype 3 pneumococcalconjugate. Schuerman L, Prymula R, Poolman J. Abstract book p 245,P010.06).

Example 6, Effectiveness of Detoxified Pneumolysin (dPly) as a ProteinCarrier to Enhance the Immunogenicity of PS 19F in Young Balb/c Mice

Groups of 40 female Balb/c mice (4-weeks old) were immunized IM at days0, 14 and 28 with 50 μl of either 4-valent plain PS or 4-valentdPly-conjugated PS, both admixed with Adjuvant C.

Both vaccine formulations were composed of 0.1 μg (quantity ofsaccharide) of each of the following PS: PS8, PS12F, PS19F and PS22F.

Anti-PS ELISA IgG levels were dosed in sera collected at day 42.

The anti-PS19F response, shown as an example in FIG. 3, was stronglyenhanced in mice given 4-valent dPly conjugates compared to miceimmunized with the plain PS. The same improvement was observed for theanti-PS8, 12F and 22F IgG responses (data not shown).

Example 7, Effectiveness of Pneumococcal Histidine Triad Protein D(PhtD) as a Protein Carrier to Enhance the Immunogenicity of PS 22F inYoung Balb/c Mice

Groups of 40 female Balb/c mice (4-weeks old) were immunized IM at days0, 14 and 28 with 50 μl of either 4-valent plain PS or 4-valentPhtD-conjugated PS, both admixed with Adjuvant C.

Both vaccine formulations were composed of 0.1 μg (quantity ofsaccharide) of each of the following PS: PS8, PS12F, PS19F and PS22F.

Anti-PS ELISA IgG levels were dosed in sera collected at day 42.

The anti-PS22F response, shown as an example in FIG. 4, was stronglyenhanced in mice given 4-valent PhtD conjugates compared to miceimmunized with the plain PS. The same improvement was observed for theanti-PS8, 12F and 19F IgG responses (data not shown).

Example 8, Immunogenicity in Elderly C57BI Mice of 13-Valent PSConjugates Containing 19A-dPly and 22F-PhtD

Groups of 30 old C57BI mice (>69-weeks old) were immunized IM at days 0,14 and 28 with 50 μl of either 11-valent PS conjugates or 13-valent PSconjugates, both admixed with Adjuvant C (see below).

The 11-valent vaccine formulation was composed of 0.1 μg saccharide ofeach of the following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD,PS6B-PD, PS7F-PD, PS9V-PD, PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (seeTable 1 and comment on 11 valent vaccine discussed under Table 2). The13-valent vaccine formulation contained in addition 0.1 μg of PS19A-dPlyand PS22F-PhtD conjugates (see Table 1 and comment on 13 valent vaccinediscussed under Table 2 [using directly-conjugated 22F]). In group 2 and4 the pneumolysin carrier was detoxified with GMBS treatment, in group 3and 5 it was done with formaldehyde. In groups 2 and 3 PhtD was used toconjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion (the construct VP147from WO 03/054007) was used. In group 6 19A was conjugated to diphtheriatoxoid and 22F to protein 0.

Anti-PS19A and 22F ELISA IgG levels were dosed in individual seracollected at day 42.

The ELISA IgG response generated to the other PS was measured in pooledsera. 19A-dPly and 22F-PhtD administered within the 13-valent conjugatevaccine formulation were shown immunogenic in old C57B mice (Table 15).The immune response induced against the other PS was not negativelyimpacted in mice given the 13-valent formulation compared to thoseimmunized with the 11-valent formulation.

TABLE 15 PS immunogenicity in old C57BI mice (post-III IgG levels) OldC57 Black mice GROUP 2 GROUP 3 GROUP 4 GROUP 5 11V 11V 11V 11V GROUP 619A-dPly 19A-dPly 19A-dPly 19A-dPly 11V GROUP 1 gmbs formol gmbs formol19A-DT 11V 22F-PhtD 22F-PhtD 22F-PhtD-E 22F-PhtD-E 22F-PD 0.1 μg/50 μl0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl ELISAAdj C Adj C Adj C Adj C Adj C Adj C 1 average 19.30 20.20 24.40 12.8012.10 13.60 Pool 3 average 6.32 4.84 5.21 6.74 2.38 2.54 Pool 4 average60.9 67.1 51.4 47.4 45.5 41.1 Pool 5 average 1.34 3.81 3.06 2.75 1.261.23 Pool 6B average 4.41 4.12 5.88 1.58 2.31 5.64 Pool 7F average 0.830.81 1.65 1.98 0.89 0.99 Pool 9V average 13.8 23.7 20.0 13.1 15.5 9.6Pool 14 average 25.73 42.96 34.12 32.53 23.97 15.60 Pool 18C average13.4 20.1 11.9 9.1 8.3 8.4 Pool 19F average 57.5 90.0 63.8 36.5 47.069.1 Pool 23F average NR NR NR NR NR NR Pool 19A GMC 0.06 0.09 0.25 0.080.23 0.19 IC 0.04-0.1 0.05-0.14 0.15-0.41 0.06-0.12 0.14-0.38 0.09-0.3 %sero 33% 47% 83% 53% 80% 73% 22F GMC NR 5.81 3.76 0.54 0.85 2.02 IC 3.2-10.6  1.8-7.9   0.3-1.1   0.4-1.7   1.2-3.4 % sero  0% 97% 90% 77%87% 97%

Example 9, Immunogenicity in Young Balb/c Mice of 13-Valent PSConjugates Containing 19A-dPly and 22F-PhtD

Groups of 30 young Balb/c mice (4-weeks old) were immunized IM at days0, 14 and 28 with 50 μl of either 11-valent PS conjugates or 13-valentPS conjugates, both admixed with Adjuvant C (see below).

The 11-valent vaccine formulation was composed of 0.1 μg saccharide ofeach of the following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD,PS6B-PD, PS7F-PD, PS9V-PD, PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (seeTable 1 and comment on 11 valent vaccine discussed under Table 2). The13-valent vaccine formulation contained in addition 0.1 μg of PS19A-dPlyand PS22F-PhtD conjugates (see Table 1 and comment on 13 valent vaccinediscussed under Table 2 [using directly-conjugated 22F]). In group 2 and4 the pneumolysin carrier was detoxified with GMBS treatment, in group 3and 5 it was done with formaldehyde. In groups 2 and 3 PhtD was used toconjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion (the construct VP147from WO 03/054007) was used. In group 6 19A was conjugated to diphtheriatoxoid and 22F to protein D.

Anti-PS19A and 22F ELISA PgG levels were dosed in individual seracollected at day 42. The ELISA IgG response generated to the other PSwas measured in pooled sera. 19A-dPly and 22F-PhtD administered withinthe 13-valent conjugate vaccine formulation were shown immunogenic inyoung Balb/c mice (Table 16). The immune response induced against theother PS was not negatively impacted in mice given the 13-valentformulation compared to those immunized with the 11-valent formulation.

TABLE 16 PS immunogenicity in young Balb/c mice (post-III IgG levels)BalbC mice GROUP 2 GROUP 3 GROUP 4 GROUP 5 11V 11V 11V 11V GROUP 619A-dPly 19A-dPly 19A-dPly 19A-dPly 11V GROUP 1 gmbs formol gmbs formol19A-DT 11V 22F-PhtD 22F-PhtD 22F-PhtD-E 22F-PhtD-E 22F-PD 0.1 μg/50 μl0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl ELISAAdj C Adj C Adj C Adj C Adj C Adj C 1 average 131.70 101.20 83.00 82.4067.90 85.50 Pool 3 average 21.85 10.38 12.53 8.83 8.73 14.98 Pool 4average 147.4 127.0 104.4 95.0 113.6 114.2 Pool 5 average 21.38 20.2918.26 18.95 18.02 23.04 Pool 6B average 1.97 4.76 3.72 2.35 1.43 1.05Pool 7F average 7.69 4.58 4.77 4.24 3.92 3.94 Pool 9V average 30.1 30.726.5 21.4 23.4 28.3 Pool 14 average 28.78 27.67 26.23 21.54 24.34 13.73Pool 18C average 53.4 52.37 46.5 57.8 47.8 75.8 Pool 19F average 186.6157.7 169.3 178.9 181.9 223.2 Pool 23F average 4.98 3.9 5.11 0.57 3.134.57 Pool 19A GMC 0.4 32.8 25.1 21.6 18.9 23.5 IC 0.2-0.6 26.4-40.720.6-30.6 17.5-26.7 15.1-23.5 19.5-28.5 % sero 93% 100% 100% 100% 100%100% 22F GMC NR 3.99 3.76 6.27 8.70 18.76 IC 1.9-8.42 1.8-8 3.8-10.45.4-13.9 15.2-23.1 % sero  0%  93% 100% 100% 100% 100%

Example 10, Immunogenicity in Guinea Pigs of 13-Valent PS ConjugatesContaining 19A-dPly and 22F-PhtD

Groups of 20 young Guinea Pigs (Hartley Strain; 5 weeks old) wereimmunized IM at days 0, 14 and 28 with 125 μl of either 11-valent PSconjugates or 13-valent PS conjugates, both admixed with Adjuvant C (seebelow).

The 11-valent vaccine formulation was composed of 0.25 μg saccharide ofeach of the following conjugates: PS1-PD, PS3-PD, PS4-PD, PS5-PD,PS6B-PD, PS7F-PD, PS9V-PD, PS14-PD, PS18C-TT, PS19F-DT and PS23F-PD (seeTable 1 and comment on 11 valent vaccine discussed under Table 2). The13-valent vaccine formulation contained in addition 0.1 μg of PS19A-dPlyand PS22F-PhtD conjugates (see Table 1 and comment on 13 valent vaccinediscussed under Table 2 [using directly-conjugated 22F]). In group 2 and4 the pneumolysin carrier was detoxified with GMBS treatment, in group 3and 5 it was done with formaldehyde. In groups 2 and 3 PhtD was used toconjugate PS 22F, in Groups 4 and 5 a PhtD_E fusion (the construct VP147from WO 03/054007) was used. In group 6 19A was conjugated to diphtheriatoxoid and 22F to protein D.

Anti-PS19A and 22F ELISA IgG levels were dosed in individual seracollected at day 42.

The ELISA IgG response generated to the other PS was measured in pooledsera.

TABLE 17 PS immunogenicity in young Balb/c mice (post-III IgG levels)Guinea pigs GROUP 2 GROUP 3 GROUP 4 GROUP 5 11V 11V 11V 11V GROUP 619A-dPly 19A-dPly 19A-dPly 19A-dPly 11V GROUP 1 gmbs formol gmbs formol19A-DT 11V 22F-PhtD 22F-PhtD 22F-PhtD-E 22F-PhtD-E 22F-PD 0.1 μg/50 μl0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl 0.1 μg/50 μl ELISAAdj C Adj C Adj C Adj C Adj C Adj C 1 average 78.00 77.21 76.15 68.7768.59 81.04 Pool 3 average 7.75 9.31 12.73 7.94 4.75 9.59 Pool 4 average130.7 94.4 132.6 166.8 85.0 101.3 Pool 5 average 109.10 117.10 110.70158.40 74.10 100.40 Pool 6B average 3.14 4.26 14.4 7.63 6.3 7.52 Pool 7Faverage 154.2 216.0 240.0 181.0 142.0 179.1 Pool 9V average 90.69 105.4598.20 93.45 54.12 73.05 Pool 14 average 71.19 77.18 46.53 59.67 38.4753.69 Pool 18C average 109.4 122.3 137.1 79.9 73.7 83.1 Pool 19F average73.9 102.5 112.2 75.5 62.3 72.1 Pool 23F average 19.19 30.74 29.44 31.5219.13 24.94 Pool 19A GMC 0.4 25.58 41.49 14.25 27.49 6.74 IC 0.24-0.6812-54.5 24.4-70.5 5.9-34.6 16.6-45.4 4-11.3 % sero 75% 100% 100% 100%100% 100% 22F GMC 0.12 2.51 3.67 45.74 30.68 96.38 IC 0.09-0.160.94-6.73 1.59-8.42 29.3-71.4 17-53.3 73.5-126.4 % sero 10%  95%  95%100% 100% 100%

Example 11: Formulations being Made and Tested

a) The following formulations are made (using the 13 valent vaccine fromtable 1 and serotype 3 from table 5—see comment on 14 valent vaccinediscussed under Table 2 [using directly-conjugated 22F or through an ADHlinker]). The saccharides are formulated with aluminium phosphate and3D-MPL as shown below.

14V 25 μg MPL 14V 10 μg MPL Sum of BAC Aluminium content -> FF Sum ofBAC Aluminium content -> FF Per Dose: Per Dose: μg ratio PS/AI μg ratioPS/AI PS carrier μg PS MPL 1/x μg AI PS carrier μg PS MPL 1/x μg AI 1 PD1 10 10 1 PD 1 10 10 3 PD 1 10 10 3 PD 1 10 10 4 PD 3 10 30 4 PD 3 10 305 PD 1 10 10 5 PD 1 10 10 6A PD 1 10 10 6A PD 1 10 10 6B PD 1 10 10 6BPD 1 10 10 7F PD 1 10 10 7F PD 1 10 10 9V PD 1 10 10 9V PD 1 10 10 14 PD1 10 10 14 PD 1 10 10 18C TT_(AH) 3 15 45 18C TT_(AH) 3 15 45 19A dPly 310 30 19A dPly 3 10 30 19F DT 3 10 30 19F DT 3 10 30 22F PhtD 3 10 3022F PhtD 3 10 30 23F PD 1 10 10 23F PD 1 10 10 BAC MPL 50/200 25  4 100BAC MPL 50/200 10  4 40 FF Aluminium Sum = 355 FF Aluminium content Sum= 295 content

b) The same saccharide formulation is adjuvanted with each of thefollowing adjuvants:

In the table herebelow the concentration of the emulsion components per500 μl dose is shown.

Adjuvant A1 Adjuvant A2 Adjuvant A3 250 μl o/w 125 μl o/w 50 μl o/wIngredients emulsion emulsion emulsion alpha 11.88 mg  5.94 mg 2.38 mgTocopherol Squalene 10.7 mg 5.35 mg 2.14 mg Tween 80 4.85 mg 2.43 mg0.97 mg Adjuvant A4 Adjuvant A5 Adjuvant A6 Adjuvant A7 250 μl o/w 250μl o/w 125 μl o/w 50 μl o/w Ingredients emulsion emulsion emulsionemulsion alpha 11.88 mg 11.88 mg 5.94 mg 2.38 mg Tocopherol Squalene10.7 mg 10.7 mg 5.35 mg 2.14 mg Tween 80 4.85 mg 4.85 mg 2.43 mg 0.97 mg3D-MPL 50 μg 25 μg 25 μg 10 μg

c) The saccharides are also formulated with two liposome basedadjuvants:

Composition of Adjuvant B1

Qualitative Quantitative (per 0.5 mL dose)

Liposomes:

DOPC 1 mg

cholesterol 0.25 mg

3DMPL 50 μg

QS21 50 μg

KH₂PO₄₁ 3.124 mg Buffer

Na₂HPO₄₁ 0.290 mg Buffer

NaCl 2.922 mg

(100 mM)

WFI q.s. ad 0.5 ml Solvent

pH 6.1

1. Total PO₄ concentration=50 mM

Composition of Adjuvant B2

Qualitative Quantitative (per 0.5 mL dose)

Liposomes:

DOPC 0.5 mg

cholesterol 0.125 mg

3DMPL 25 μg

QS21 25 μg

KH₂PO₄₁ 3.124 mg Buffer

Na₂HPO₄₁ 0.290 mg Buffer

NaCl 2.922 mg

(100 mM)

WFI q.s. ad 0.5 ml Solvent

pH 6.1

d) The saccharides are also formulated with Adjuvant C (see above forother compositions where this adjuvant has been used):

Qualitative Quantitative (per 0.5 mL dose)

Oil in water emulsion: 50 μl

squalene 2.136 mg

α-tocopherol 2.372 mg

Tween 80 0.97 mg

cholesterol 0.1 mg

3DMPL 50 μg

QS21 50 μg

KH₂PO₄ ₁ 0.470 mg Buffer

Na₂HPO₄₁ 0.219 mg Buffer

NaCl 4.003 mg

(137 mM)

KCl 0.101 mg

(2.7 mM)

WFI q.s. ad 0.5 ml Solvent

pH 6.8

Example 12, Impact of Conjugation Chemistry on 22F-PhtD ConjugateImmunogenicity in Balb/c Mice

Groups of 30 female Balb/c mice were immunised by the intramuscular (IM)route at days 0, 14 and 28 with 13-valent PS formulations containing PS1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F (dose: 0.3 μgsaccharide/conjugate for PS 4, 18C, 19A, 19F and 22F and 0.1 μgsaccharide/conjugate for the other PS).

PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria Toxoid, 19A toformol-detoxified Ply, 22F to PhtD and the other PS to PD.

Two formulations, constituted of either 22F-PhtD prepared by direct CDAPchemistry or 22F-AH-PhtD (ADH-derivitized PS), were compared. SeeExample 2, Table 1 and comment under Table 2 for characteristics of 13valent vaccine made either with 22F directly conjugated or via an ADHspacer. The vaccine formulations were supplemented with adjuvant C.

Anti-PS22F ELISA IgG levels and opsono-phagocytosis titres were measuredin sera collected at day 42.

22F-AH-PhtD was shown much more immunogenic than 22F-PhtD in terms ofboth IgG levels (FIG. 5) and opsono-phagocytic titres (FIG. 6).

Example 13, Impact of New Adjuvants on Immunogenicity of StreptococcusPneumoniae Capsule PS Conjugates

Groups of 40 female Balb/c mice were immunised by the IM route at days0, 14 and 28 with 13-valent PS formulations containing PS 1, 3, 4, 5,6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F (dose: 0.3 μg/conjugate forPS 4, 18C, 19A, 19F and 22F and 0.1 μg/conjugate for the other PS).

PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria Toxoid, 19A toformol-detoxified Ply, 22F to PhtD and the other PS to PD. See Example2, Table 1 and comment under Table 2 for characteristics of 13 valentvaccine made with 22F directly conjugated.

Four formulations, supplemented with either AlPO₄, adjuvant A1, adjuvantA4 or adjuvant A5, were compared.

Anti-PS, Ply, PhtD and PD ELISA IgG levels were measured in seracollected at day 42 and pooled per group. The following ratio wascalculated for each antigen: IgG level induced with the new adjuvanttested/IgG level induced with AlPO₄.

All the new adjuvants tested improved at least 2-fold the immuneresponses to 13-valent conjugates compared to the classical AlPO₄formulation (FIG. 7).

Example 14, Protective Efficacy of a PhtD/Detoxified Ply Combo in aPneumococcal Monkey Pneumonia Model

Groups of 6 Rhesus monkeys (3 to 8 years-old), selected as those havingthe lowest pre-existing anti-19F antibody levels, were immunizedintramuscularly at days 0 and 28 with either 11-valent PS conjugates(i.e. 1 μg of PS 1, 3, 5, 6B, 7F, 9V, 14 and 23F, and 3 μg of PS 4, 18Cand 19F [of saccharide]) or PhtD (10 μg)+formol-detoxified Ply (10 μg)or the adjuvant alone.

PS 18C was conjugated to Tetanus Toxoid, 19F to Diphteria Toxoid and theother PS to PD. See Example 2, Table 1 and comment under Table 2 forcharacteristics of 11 valent vaccine. All formulations were supplementedwith adjuvant C.

Type 19F pneumococci (5·10⁸ cfu) were inoculated in the right lung atday 42. Colonies were counted in broncho-alveolar lavages collected atdays 1, 3 and 7 post-challenge.

The results were expressed as the number of animals per group eitherdead, lung colonized or cleared at day 7 after challenge.

As shown in FIG. 8, a good protection close to statistical significance(despite the low number of animals used) was obtained with 11-valentconjugates and the PhtD+dPly combo (p<0.12, Fisher Exact test) comparedto the adjuvant alone group.

Example 15, Impact of Conjugation Chemistry on the Anti-PhtD AntibodyResponse and the Protective Efficacy Against a Type 4 Challenge Inducedby 22F-PhtD Conjugates

Groups of 20 female OF1 mice were immunised by the intramuscular routeat days 0 and 14 with 3 μg of either 22F-PhtD (prepared by direct CDAPchemistry) or 22F-AH-PhtD (ADH-derivitized PS), or the adjuvant alone.Both monovalent 22F conjugates were made by the processes of Example 2(see also Table 1 and Table 2). Each formulation was supplemented withadjuvant C.

Anti-PhtD ELISA IgG levels were measured in sera collected at day 27.

Mice were challenged intranasally with 5·10⁶ cfu of type 4 pneumococciat day 28 (i.e. a pneumococcal serotype not potentially covered by thePS present in the vaccine formulation tested). The mortality induced wasmonitored until day 8 post-challenge.

22F-AH-PhtD induced a significantly higher anti-PhtD IgG response andbetter protection against type 4 challenge than 22F-PhtD.

1-11. (canceled)
 12. An immunogenic composition for infants comprising amultivalent Streptococcus pneumoniae vaccine comprising at least 13capsular saccharide conjugates from different S. pneumoniae serotypes,wherein the composition comprises conjugates of S. pneumoniae capsularsaccharides 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F and 23F,wherein serotype 22F is directly conjugated to a carrier protein that isCRM197 or PhtD, and wherein serotype 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C,19A, 19F and 23F are conjugated to a carrier protein independentlyselected from TT, DT, CRM197, fragment C of TT, PhtD, PhtDE fusions,detoxified pneumolysin and protein D, wherein the average size (Mw) ofthe 22F saccharide is between 50 and 800 kDa and wherein the ratio ofcarrier protein to S. pneumoniae (22F) saccharide is between 4:1 and 1:1(w/w).
 13. The immunogenic composition according to claim 12 wherein 2different carrier proteins are separately conjugated to at least 2different S. pneumoniae capsular saccharide serotypes.
 14. Theimmunogenic composition according to claim 12 wherein at least one S.pneumoniae capsular saccharide is conjugated to the carrier protein viaa linker.
 15. The immunogenic composition according to claim 14 whereinthe linker is Adipic Acid Dihydrazide (ADH).
 16. The immunogeniccomposition according to claim 14 wherein the linker is attached to thecarrier protein by carbodiimide chemistry using1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDAC).
 17. Theimmunogenic composition according to claim 12 comprising a 22Fsaccharide conjugate, wherein the ratio of carrier protein to 22Fsaccharide is between 1:1 and 3.5:1, 1.2:1 and 3:1, or 1.5:1 and 2.5:1(w/w).
 18. The immunogenic composition according to claim 17, whereinthe ratio of carrier protein to 22F saccharide is between 1:2 and 2.5:1(w/w).
 19. The immunogenic composition according to claim 12, wherein 9or more of the conjugates have a ratio of carrier protein to saccharidethat is greater than 1:1 (w/w).
 20. The immunogenic compositionaccording to claim 12 comprising a 22F saccharide conjugate, wherein theaverage size of the 22F saccharide is above 100 kDa.
 21. The immunogeniccomposition according to claim 12 comprising a 22F saccharide conjugate,wherein the average size of the 22F saccharide is between 110 and 700kDa, 110-300, 120-200, 130-180, or 150-170 kDa.
 22. The immunogeniccomposition according to claim 12 comprising conjugates of S. pneumoniaecapsular saccharides wherein the average size of the saccharides is50-1600, 80-1400, 100-1000, 150-500, or 200-400 kDa.
 23. The immunogeniccomposition according to claim 12 which further comprises one or moreunconjugated or conjugated S. pneumoniae proteins.
 24. The immunogeniccomposition according to claim 19 wherein said one or more unconjugatedor conjugated S. pneumoniae proteins is selected from Poly HistidineTriad family (PhtX), Choline Binding Protein (CbpX), CbpX truncates,LytX family, LytX truncates, CbpX truncate-LytX truncate chimericproteins, detoxified pneumolysin (Ply), PspA, PsaA, Sp128, Sp101, Sp130,Sp125 and Sp133.
 25. The immunogenic composition according to claim 12which further comprises an adjuvant.
 26. The immunogenic compositionaccording to claim 25 wherein the adjuvant comprises (per 0.5 mL dose)100-750, 200-500, or 300-400 μg Al aluminium phosphate.